Antibodies to ARF-p19

ABSTRACT

The INK4A (MTS1, CDKN2) gene encodes a specific inhibitor (InK4a-p16) of the cyclin D-dependent kinases CDK4 and CDK6. InK4a-p16 can block these kinase from phosphorylating the retinoblastoma protein (pRb), preventing exit from the G 1  phase of the cell cycle. Deletions and mutations involving the gene encoding InK4a-p16, INK4A, occur frequently in cancer cells, implying that INK4a-p16, like pRb, suppresses tumor formulation. However, a completely unrelated protein (ARF-p19) arises in major part from an alternative reading frame of the mouse INK4A gene. Expression of an ARF-p19 cDNA (SEQ ID NO:1) in rodent fibroblasts induces both G1 and G2 phase arrest.

This application is a Divisional of Application Ser. No: 09/247,154filed Feb. 9, 1999, which is a Continuation of application Ser. No.:08/954,470, filed Oct. 20, 1997, now issued U.S. Pat. No. 5,876,965having an issue Date of Mar. 2, 1999, which is a Divisional Applicationof application Ser. No. 08/534,975 Filed on Sep. 27, 1995, now issuedU.S. Pat. No. 5,723,313 having an issue Date of Mar. 3, 1998, thedisclosures of which are hereby incorporated herein by reference intheir entireties. Applicants claim the benefits of these Applicationsunder 35 U.S.C. §120.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with U.S. Government support under Cancer CenterCORE grant 5P30CA21765-18, awarded by the National Cancer Institute. TheU.S. Government has certain rights in this invention. Support for thisinvention was also provided by the Howard Hughes Medical Institute andthe American Lebanese Syrian Associated Charities of St. Jude Children'sResearch Hospital.

FIELD OF THE INVENTION

This invention relates to cancer detection and treatment and, moreparticularly, to a novel protein called “p19^(ARF) protein,” “p19^(ARF)”or simply “ARF-p19” that is involved in regulation of the eukaryoticcell cycle. Protein ARF-p19 is encoded by a nucleic acid derived fromthe gene, INK4A, which also encodes an inhibitor of D-typecyclin-dependent kinases called “p16^(Ink4a) protein,” “p16^(InK4a)” orsimply “InK4a-p16.”

Transcripts encoding InK4a-p16 originate from a first promoter, E1α; thepresent invention is based on the observation that some INK4Atranscripts initiate from a second promoter, E1β, and contain anAlternative Reading Frame, ARF, which overlaps the InK4a-p16 readingframe to some degree. ARF transcripts direct the production of a proteinthat has ARF-p19 amino acid sequences instead of the previously-knownInK4a-p16 sequences. Like InK4a-p16, ARF-p19 regulates the eukaryoticcell cycle. When overexpressed, ARF-p19 inhibits cells from proceedingpast both the G1 and G2 phases of the cell cycle. However, themechanism(s) by which ARF-p19 acts are unlike those of InK4a-p16, whichacts by directly and specifically interacting with CDK (cyclinD-dependent kinase) proteins and thus preventing CDK-cyclin Dinteractions.

In addition to (1) ARF-p19 proteins, this invention further relates to(2) nucleic acids that encode ARF-p19 isolated from mice, humans andother mammals; (3) antibodies that specifically bind ARF-p19 protein orpolypeptides derived therefrom; (4) methods for detecting one or morenucleic acids encoding ARF-p19, or alterations in such nucleic acids;(5) methods for producing ARF-p19 proteins using nucleic acids thatencode ARF-p19; (6) purified ARF-p19 proteins, or fusion proteinsderived from the joining of an ARF-p19 polypeptide sequence with asecond polypeptide sequence; (7) methods of treating cancer usingpurified ARF-p19 proteins or fusion proteins derived therefrom; (8)methods of inducing cell cycle arrest using ARF-pi9 proteins or nucleicacids encoding ARF-p19 proteins; (9) methods for detecting ARF-p19proteins using antibodies that specifically bind ARF-p19 proteins; (10)methods of selectively killing cells having uncontrolled growth usingantibodies that specifically bind ARF-p19 proteins, or conjugatesderived from such antibodies; (11) methods of stimulating cell growthusing antibodies that specifically bind ARF-p19 proteins, or fragmentsderived from such antibodies; and (12) transgenic non-human animals thathave a genetically engineered alteration in one or more nucleic acidsencoding ARF-p19 proteins but which express normal levels of wild-typeInK4a-p16 protein, or which overexpress human ARF-p19 or mutant forms ofARF-p19.

BACKGROUND OF THE INVENTION

Neoplasia, the pathological process by which tumors develop, necessarilyinvolves unregulated, or at best misregulated, cellular growth anddivision. The molecular pathways that regulate cellular growth mustinevitably intersect with those that regulate the cell cycle. The cellcycle consists of a cell division phase and the events that occur duringthe period between successive cell divisions, known as interphase.Interphase is composed of successive G1, S, and G2 phases, and normallycomprises 90% or more of the total cell cycle time. Most cell componentsare made continuously throughout interphase; it is therefore difficultto define distinct stages in the progression of the growing cell throughinterphase. One exception is DNA synthesis, since the DNA in the cellnucleus is replicated only during a limited portion of interphase. Thisperiod is denoted as the S phase (S=synthesis) of the cell cycle. Theother distinct stage of the cell cycle is the cell division phase, whichincludes both nuclear division (mitosis) and the cytoplasmic division(cytokinesis) that follows. The entire cell division phase is denoted asthe M phase (M=mitotic). This leaves the period between the M phase andthe start of DNA synthesis, which is called the G1 phase (G=gap), andthe period between the completion of DNA synthesis and the next M phase,which is called the G2 phase (Alberts, B. et al., Molecular Biology ofthe Cell, Garland Publishing, Inc., New York & London (1983), pages611-612.).

Progression through different transitions in the eukaryotic cell cycleis positively regulated by a family of master enzymes, thecyclin-dependent kinases (reviewed by Sherr, C. J., Cell 73:1059-1065(1993)). These holoenzymes are composed of two proteins, a regulatorysubunit (the cyclin), and an associated catalytic subunit (the actualcyclin-dependent kinase or CDK), the levels of which vary with differentphases of the cell cycle (Peters, G., Nature 371:204-205 (1994)). Bothcyclins and CDKs represent molecular families that encompass a varietyof genetically related but functionally distinct proteins. Generally,different types of cyclins are designated by letters (i.e., cyclin A,cyclin B, cyclin D, cyclin E, etc.); CDKs are distinguished by numbers(CDK1, CDK2, CDK3, CDK4, CDK5, etc.; CDK1 is a.k.a. CDC2).

CDK-cyclin D complexes regulate the decision of cells to replicate theirchromosomal DNA (Sherr, Cell 73:1059-1065 (1993)). As cells enter thecycle from quiescence, the accumulation of CDK-cyclin D holoenzymesoccurs in response to mitogenic stimulation, with their kinaseactivities being first detected in mid-G1 phase and increasing as cellsapproach the G1/S boundary (Matsushime et al., Mol. Cell. Biol.14:2066-2076 (1994); Meyerson and Harlow, Mol. Cell. Biol. 14:2077-2086(1994)). The cyclin D regulatory subunits are highly labile, andpremature withdrawal of growth factors in G1 phase results in a rapiddecay of CDK-cyclin D activity that correlates with the failure to enterS phase. In contrast, removal of growth factors late in G1 phase,although resulting in a similar collapse of CDK-cyclin D activity, hasno effect on further progression through the cell cycle (Matsushime etal., Cell 65:701-713 (1991)). Microinjection of antibodies to cyclin D1into fibroblasts during G1 prevents entry into the S phase, butinjections performed at or after the G1→S transition are without effect(Baldin et al., Genes & Devel. 7:812-821 (1993); Quelle et al., Genes &Devel. 7:1559-1571 (1993)). Therefore, CDK-cyclin D complexes executetheir critical functions at a late G1 checkpoint, after which cellsbecome independent of mitogens for completion of the cycle.

In mammals, cells enter the cell cycle and progress through G1 phase inresponse to extracellular growth signals which trigger thetranscriptional induction of D-type cyclins. The accumulation of Dcyclins leads to their association with two distinct catalytic partners,CDK4 and CDK6, to form kinase holoenzymes. Several observations arguefor a significant role of the cyclin D-dependent kinases inphosphorylating the retinoblastoma protein, pRb, leading to the releaseof pRB-associated transcription factors that are necessary to facilitateprogression through the G1→S transition. First, CDK-cyclin D complexeshave a distinct substrate preference for pRb but do not phosphorylatethe canonical CDK substrate, histone H1 (Matsushime et al., Cell71:323-334 (1992); Matsushime et al., Mol. Cell. Biol. 14:2066-2076(1994); Meyerson and Harlow, Mol. Cell. Biol. 14:2077-2086 (1994)).Their substrate specificity may be mediated in part by the ability ofD-type cyclins to bind to pRb directly, an interaction which isfacilitated by a Leu-X-Cys-X-Glu pentapeptide that the D cyclins sharewith DNA oncoproteins that also bind pRb (Dowdy et al., Cell 73:499-511(1993); Ewen et al., Cell 73:487-497 (1993); Kato et al., Genes & Devel.7:331-342 (1993)). Second, cells in which pRb function has beendisrupted by mutation, deletion, or after transformation by DNA tumorviruses are no longer inhibited from entering S phase by microinjectionof antibodies to D cyclin, indicating that they have lost theirdependency on the cyclin D-regulated G1 checkpoint (Lukas et al., J.Cell. Biol. 125:625-638 (1994); Tam et al., Oncogene 9:2663-2674(1994)). However, introduction of pRb into such cells restores theirrequirement for cyclin D function (Lukas et al., J. Cell. Biol.125:625-638 (1994)). Third, pRb-negative cells synthesize elevatedlevels of a 16 kDa polypeptide inhibitor of CDK4, “p16^(InK4a)” (a.k.a.“InK4a-p16” or simply “p16”), which is a member of a recently discoveredclass of cell cycle regulatory proteins (Nasmyth and Hunt, Nature366:634-635 (1993); Peters, G., Nature 371:204-205 (1994)) and which isfound in complexes with CDK4 at the expense of D-type cyclins during G1phase (Bates et al., Oncogene 9:1633-1640 (1994); Serrano et al., Nature366:704-707 (1993); Xiong et al., Genes & Devel. 7:1572-1583 (1993)).The fact that such cells cycle in the face of apparent CDK4 inhibitionagain implies that D-type cyclins are dispensable in the Rb-negativesetting.

The InK4 gene family (“InK4” signifies Inhibitors of CDK4) is known toinclude at least three other low molecular weight polypeptides,InK4b-p15, induced in human epithelial cells treated by transforminggrowth factory-β (TGF-β) (Hannon, G. J., and Beach, D., Nature371:257-261 (1994)), InK4d-p19 (Hirai, H., et al., Mol. Cell. Biol.15:2672-2681 (1995)) and InK4c-p18 (Guan et al., Genes & Develop.8:2939-2952 (1994); Hirai, H., et al., Mol. Cell. Biol. 15:2672-2681(1995)). InK4d-p19 and InK4c-p18 are described in detail in Ser. No.08/384,106, filed Feb. 6, 1995, which is hereby incorporated byreference.

Members of the InK4 family are typically composed of repeated ankyrinmotifs, each of about 32 amino acids in length. All known members of theInK4 family act to specifically inhibit enzymatic activities of D-typecyclin-dependent kinases such as CDK4 and CDK6. Unlike other universalCDK inhibitors, such as p21^(Cip1/Waf1) (E1-Deiry et al., Cell75:817-825 (1993); Gu et al., Nature 366:707-710 (1993); Harper et al.,Cell 75:805-816 (1993); Xiong et al., Nature 366:701-704 (1993)) andp27^(Kip1) (Polyak et al., Genes & Devel. 8:9-22 (1994); Polyak et al.,Cell 78:59-66 (1994); Toyoshima and Hunter, Cell 78:67-74 (1994)), theInK4 proteins selectively inhibit the activities of CDK4 and CDK6, butdo not inhibit the activities of other CDKs (Guan et al., Genes & Devel.8:2939-2952 (1994); Hannon and Beach, Nature 371:257-261 (1994); Serranoet al., Nature 366:704-707 (1993)).

Like many CDK inhibitors (CKIs) (Nasmyth and Hunt, Nature 366:634-635(1993)), InK4 family members negatively regulate progression through themammalian cell cycle, in part in response to anti-proliferativeextracellular signals. The InK4 proteins, by inhibiting the activitiesof a specific class of the D-type cyclin-dependent kinases (i.e., CDK4and/or CDK6), arrest cell cycle progression in G1 phase and thus preventcells from replicating their chromosomal DNA. Thus, in contradistinctionto the positive regulation of D-type cyclin synthesis by growth factors,extracellular inhibitors of G1 progression can negatively regulate theactivity of D-type cyclin-dependent kinases by inducing InK4 proteins.

RELATED ART

Mullis et al., U.S. Pat. No. 4,965,188 (Oct. 23, 1990), describe methodsfor amplifying nucleic acid sequences using the polymerase chainreaction (PCR).

Beach, published PCT patent application WO 92/20796 (Nov. 26, 1992),describes genes encoding D cyclins and uses thereof.

Berns, U.S. Pat. No. 5,174,986 (Dec. 29, 1992), describes methods fordetermining the oncogenic potential of chemical compounds using atransgenic mouse predisposed to develop T-cell lymphomas.

Crissman et al., U.S. Pat. No. 5,185,260 (Feb. 9, 1993), describemethods for distinguishing and selectively killing transformed(neoplastic) cells using synthetic G1 kinase inhibitors.

Stone, S., et al., Cancer Research 55:2988-2994 (1995), describe twocDNAs derived from the human INK4A gene, including an “αform” encodingInK4a-p16 and β“, form” that includes an open reading frame (designated“ORF 2”) that overlaps the reading frame encoding the ARF-p19 proteindescribed herein. Stone et al. state that it “is unknown if ORF 2encodes a protein” (legend to FIG. 1, page 2990) and indicate that “ORF2 has not been selectively maintained and probably does not encode aprotein” (page 2989, column 2, lines 20-21).

Mao, L., et al., Cancer Research 55:2995-2997 (1995), describe twotranscripts and corresponding cDNAs derived from the human INK4A gene,designated “p16” and “p16β.” The p16 transcript is stated to encode theInK4a-p16 protein, while the p16β transcript is stated to contain a“theoretical open reading frame” (page 1996, column 1, line 47) that isnot further defined, and suggest this sequence “probably represents anuntranslated open reading frame” (page 2997, column 2, lines 9-10). Maoet al. state that the in vitro transcription and translation (TNT)product of the p16β cDNA is recognized by an antibody to InK4a-p16polypeptide sequences (page 2997, column 1, lines 6), suggesting thatthe p16β transcript encodes an amino-terminal truncated InK4a-p16polypeptide rather than a protein having, as ARF-p19 does, an amino acidsequence unrelated to that of InK4a-p16. However, Mao et al. also statethat, using InK4a-p16 antiserum, they are unable to identify anamino-terminal truncated p16β protein in cell lines (page 2997, column2, lines 1-2). Thus, Mao et al. are silent regarding the ARF-p19 proteindescribed herein.

SUMMARY OF THE INVENTION

The present invention relates to the discovery in mammalian cells of anovel cell cycle regulatory protein, having a predicted molecular massof 19 kDa, here designated “ARF-p19 protein” or more simply “ARF-p19”.In particular, the invention relates to ARF-p19 proteins isolated fromcells derived from a mouse or a human. Although derived from the geneencoding the previously-known InK4a-p16 protein, ARF-p19 arises bydifferential transcription and translation of InK4a-p16 sequences. Thatis, ARF-p19 is encoded by an alternative reading frame (ARF) and has anamino acid sequence (SEQ ID NO:2; SEQ ID NO:4) that is wholly unrelatedto that of InK4a-p16. Surprisingly, however, ARF-p19 protein functionsto regulate the cell cycle in a similar but less specific manner than,and by a mechanism distinct from that of, InK4a-p16 protein.

Thus, one aspect of the invention is directed to methods of using theARF-p19 proteins of the invention to inhibit the growth of cancer cellsand/or to prevent cancer cells from replicating their chromosomal DNA.Both InK4-p16 and InK4-p15 appear to act as tumor suppressors (Noburi,T. et al., Nature 368:753-756 (1994); Karnb, A. et al., Science264:436440 (1994)). The genes encoding p16 and p15 map in a tandem arrayto the short arm of human chromosome 9 within a region that isfrequently deleted in cancer cells, and the resulting loss of theiranti-proliferative functions can contribute to tumorigenesis (Noburi etal., Nature 368:753-756 (1994); Okuda, T., et al., Blood 85:2321-2330(1995)). The novel ARF-p19 protein described herein (1) plays a role inpreventing the G1→S and G2→M phase transitions in normal mammaliancells, and (2) if having reduced or altered activity due to one or moremutations affecting the alternative reading frame encoding ARF-p19,could contribute to oncogenesis in some cancers, even if such mutationshave no effect on the reading frame encoding InK4a-p16.

In another aspect, the invention provides nucleic acid sequencesencoding ARF-p19 polypeptides from mice, humans and other mammals. Thenucleic acid sequences of the invention may be expressed in the form ofisolated nucleic acids, such as cDNA clones, genomic DNA clones, MRNAtranscribed from either cDNA or genomic DNA clones, syntheticoligonucleotides, and/or synthetic amplification products resulting fromPCR, and may be single-stranded or double-stranded.

In a related aspect, the invention provides methods for detectingnucleic acids encoding wild-type or mutant ARF-p19 proteins using thenucleic acid sequences of the invention described above. The detectionof point mutations, deletions of, or other mutations in, the readingframe encoding ARF-p19 is predictive of a predisposition to, ordiagnostic of, certain types of cancer.

In another related aspect, the DNA molecules of the invention describedabove may be cloned into expression vectors and placed in an appropriatehost in order to produce ARF-p19 proteins or fusion proteins containingARF-p19 polypeptide sequences. When placed in an animal that has cancer,this aspect of the invention relates to gene therapy for certain typesof cancers.

In another aspect, the invention provides antibody compositions thatbind specifically to ARF-p19 proteins and/or polypeptides derivedtherefrom. The antibody compositions of the invention may be polyclonal,monoclonal, or monospecific. Although all of the antibody compositionsof the invention bind specifically to ARF-p19, some compositions bind toa specific epitope of ARF-p19 and thereby inhibit a specific function ofARF-p19.

In a related aspect, the invention provides methods for detectingARF-p19 proteins using the antibody compositions described above. Thedetection of reduced amounts of, or altered forms of, ARF-p19 proteinsis predictive of a predisposition to, or diagnostic of, certain types ofcancer.

In another aspect, the invention provides transgenic non-human animalswhich have one or more mutations in the endogenous reading frameencoding ARF-p19, wherein said mutation results in the production of amutant ARF-p19 protein or results in a loss of ARF-p19 expression butdoes not significantly affect the InK4a-p16 gene product or expressionthereof. Additionally or alternatively, the transgenic non-human animalsof the invention express a human wild-type or mutant ARF-p19. Because ofthe transgene(s) introduced into the genome of the non-human animals ofthe invention, the animals have a reduced and/or altered ARF-p19activity compared to wild-type animals, and consequentially developscertain types of cancers, particularly melanomas, in a reproducible andthus predictable manner.

In a related aspect, compositions are evaluated for their potential toenhance or inhibit certain types of cancers, particularly melanomas,using the transgenic non-human animals of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the sequence of a murine cDNA molecule (SEQ ID NO:1)homologous to human INK4A β transcripts and the sequence of thepolypeptide, ARF-p19 (SEQ ID NO:2), encoded thereby; the partialcarboxy-terminal amino acid sequence of InK4a-p16 (SEQ ID NO:5), encodedby exons 2 and 3 in INK4A α transcripts, is also indicated. Sequencesfrom exon 1β (nucleotides 1-232) are spliced to exon 2 to create an openreading frame capable of encoding a novel 169 amino acid protein(ARF-p19). The initiator codon for ARF-p19 occurs at nucleotides 43-45,and a putative UAG stop codon is found at nt 550-552. Nucleotides436-438 (double underlined CCA) are replaced by a TGA termination codonin corresponding position in the human INK4A gene (Serrano et al.,Nature 366:704-707 (1993)); accordingly, the human ARF-p19 protein istruncated relative to the murine ARF-p19 protein. Unrelated sequencesfrom exon 1α (not shown) are spliced to the same exon 2 acceptor site toopen another reading frame that encodes InK4a-p16. Exon 2-codedInK4a-p16 amino acid sequences are shown below those of ARF-p19. Thecarboxyl terminus of mouse InK4a-p16 is 20 residues longer than thehuman polypeptide (Quelle et al., Oncogene 11:635-645 (1995)), with thelast four amino acids of the latter encoded by a third exon (Kamb, A. etal., Science 264:436-440 (1994a)). The location of residuescorresponding to the primers used to specifically amplify β transcriptsby RT-PCR (FIG. 2) are underlined.

FIG. 2 (panels A-D) shows the results of RT-PCR assays of INK4A α and βmRNA transcripts in mouse tissues and the mouse MEL cell line.Equivalent quantities of RNA from the indicated tissues were amplifiedin parallel by RT-PCR using 5′ primers specific for α or β transcriptsand a common 3′ primer (FIG. 1). Products from β (panels A, B) and α(panels C, D) RT templates were hybridized with specific exon 1β (panelA), exon 1α (panel C), or exon 2 probes (panels B, D). Autoradiographicexposure times were 2 hrs.

FIG. 3 shows the results of immunoassays using antibodies to thecarboxyl-terminal portion of ARF-p19 (p19 antiserum) or antibodies toInK4a-p16 (p16 antiserum) and the detection of InK4a-p16 (p16), ARF-p19(p19) or ARF-p19 tagged with hemagglutinin (HA-p19). cDNAs encodingInK4a-p16, ARF-p19, and HA-tagged ARF-p19 (as indicated below the panel)were transcribed and translated in vitro. Proteins labeled with[³⁵S]-Methionine, normalized for equal input of radioactivity, wereprecipitated with nonimmune rabbit serum (NRS) or with antisera toInK4a-p16 or ARF-p19 (as indicated at the top) and separated ondenaturing gels. The positions of marker proteins of known molecularmass are indicated at the right. is FIG. 4 (panels A-C) shows theresults of immunoassays using antibodies specific for ARF-p19,InK4a-p16, or hemagglutinin (HA). Cell lysates (indicated at the bottomof panel C) were divided into three equal aliquots, separated ondenaturing gels, and immunoblotted with antibodies specific for ARF-p19(panel A), HA (panel B) or InK4a-p16 (panel C). The cell lines indicatedon the bottom of the Figure (Sf9=insect cells in which baculoviralexpression occurs; N3T3-d=NIH-3T3 cells genetically engineered tooverexpress cyclin D1; B3T3=derivative of Balb-3T3 cells; MEL=mouseerythroleukemia cells) were either uninfected (“none”) or infected withappropriate expression constructs expressing ARF-p19 (“p19”), ARF-p19tagged with hemagglutinin (“HA-p19”), or InK4a-p16 (“p16”). Cells wereinfected for 48 hrs before lysis with control vectors lacking inserts(lanes 1, 3, and 7) or containing the indicated cDNAs (top, panel A).The positions of marker proteins are shown at the left and positions ofARF-p19 or InK4a-p16 at the right and by arrowheads in panels A and B.Blots were developed using enhanced fluorography (exposure time, 3secs), allowing only approximate comparisons of signal intensitiesbetween the different panels.

FIG. 5 (panels A-C) shows the localization of ARF-p19 andp19-hemagglutinin fusion proteins to cellular nuclei. Cytospinpreparations of NIH-3T3 cells infected for 48 hrs with a vector encodingHA-tagged ARF-p19 were fixed and stained with antiserum to ARF-p19(panel A), anti-ARF-p19 plus cognate peptide (panel B), or anti-HA serum(panel C). Matched exposures are shown at 600X magnification. Theaddition of polypeptides having ARF-p19 sequences blocks the signalproduced by the antibodies specific for ARF-p19 (panel B).

FIG. 6 summarizes mutant amino acid residues in ARF-p19 predicted frommutations in the gene that encodes both p16-InK4a and ARF-p19 compiledfrom data from primary tumors, xenografts, and established cell lines.The majority of INK4A mutations so far described target the 5′ portionof INK4A exon 2 (Hirama and Koeffler, Blood 86:841-854 (1995)), whichencodes portions of both InK4a-p16 and ARF-p19. Comparison of the mouse(upper) and human (lower) ARF-p19 amino acid sequences defines conservedresidues (bold type). Residues in the human gene that have sustainedmutations in cancer cells are doubly underlined and the mutant aminoacids are indicated below them. Mutations that are silent with regard tothe InK4a-p16 coding frame but which are predicted to affect the primarystructure of ARF-p19 are indicated by asterisks (e.g., P71T*).Superscripts note multiple substitutions of the same residue (e.g. G68L³was independently observed in 3 cases), and closed squares definemicrodeletions plus frame shifts. No nonsense mutations were found. Allmutations were detected in sporadic cancers except for R114L (G101W inp16), which has been genetically implicated in familial melanoma in 3 of9 kindreds (Hussussian et al., Nature Genet 8:15-21 (1994); Kamb et al.,Nature Genet 8:22-26 (1994b)). Known sequence polymorphisms have beenexcluded. The remaining data were taken from Caldas et al., Nature Genet8:27-32 (1994), Hayashi et al., Biochem. Biophrys. Res Commun.202:1426-1430 (1994), Kanb, A. et al., Science 264:436-440 (1994), Moriet al., Cancer Res. 54:3396-3397 (1994), Ohta et al., Cancer Res54:5269-5272 (1994), and Zhang et al., Cancer Res 54:5050-5053 (1994).Numbering of InK4a-p16 amino acid sequences in the text is based on thecorrected N-terminus (Hannon and Beach, Nature 371:257-261 (1994)) whichincludes 8 residues beyond those originally identified (Serrano et al.,Nature 366:704-707 (1993)).

FIG. 7 shows the sequence of a human cDNA molecule (3; see also Mao etal., Cancer Research 55:2995-2997 91995)) corresponding to human INK4A βtranscripts and the sequence of the polypeptide, ARF-p19 (SEQ ID NO:4,denoted “arf” in the Figure), which is (as described herein) encodedthereby. The partial carboxyl-terminal amino acid sequence of humanInK4a-p16 (“p16”) is also indicated.

DETAILED DESCRIPTION OF THE DISCLOSURE Terms and Symbols

For purposes of this disclosure, the following abbreviations anddefinitions are used herein unless otherwise indicated.

The following list indicates the correspondence between the one-letteramino acid code (used in FIGS. 1, 6 and 7 and Example 6) and thethree-letter amino acid code (used elsewhere herein, in accordance with37 C.F.R. § 1.822, revised as of Jul. 1, 1994):

A = Ala C = Cys D = Asp E = Glu F = Phe G = Gly H = His I = Ile K = LysL = Leu M = Met N = Asn P = Pro Q = Gln R = Arg S = Ser T = Thr V = ValW = Trp Y = Tyr

ABBREVIATIONS CDK = cyclin-dependent kinase (protein); cdk = gene cDNA =complementary deoxyribonucleic acid DNA = deoxyribonucleic acid DMEM =Dulbecco's modified Eagle's medium EDTA = ethylenediamine tetraaceticacid ES = embryonic stem FISH = fluorescent in situ hybridization InK =inhibitor of CDK (protein); INK = gene kb = kilobase(s) kDa =kilodalton(s) MEL = mouse erythroleukemia (cell line) nt = nucleotide(s)PBS = phosphate-buffered saline PCR = polymerase chain reaction pRB =retinoblastoma protein RT = reverse transcriptase SDS = sodium dodecylsulfate Sf9 = Spodoptera frugiperda (cell line) Tg = transgenic TK =thymidine kinase

Throughout the disclosure, abbreviations for nucleodide residues presentin nucleic acid sequences are as described in 37 C.F.R. § 1.822, revisedas of Jul. 1, 1994.

Glossary

Amino acid sequence: The sequence of a polypeptide given in the order offrom amino terminal (N-terminal), to carboxyl terminal (C-terminal),Synonymous with “polypeptide sequence, ” “peptide sequence,” “proteinsequence,” or “primary protein sequence.”

Animal: (1) Excludes human beings, individually and collectively, in allstages of development, including embryonic and fetal stages, unlessotherwise indicated; and (2) includes all other vertebrate animals,including an individual animal in any stage of development, includingembryonic and fetal stages. “Non-human animal” has the same meaning as“animal.”

Animal model: A non-human animal that faithfully mimics a human diseaseand in which potential therapeutic compositions or potentially harmfulcompositions may be evaluated for their effect on the disease.

Antibody: A protein molecule synthesized by a B-cell upon exposure toantigen capable of combining specifically with that antigen. Synonymouswith immunoglobulin (Ig).

Antibody, polyclonal: A composition that comprises an assortment ofdifferent antibodies that all recognize a particular antigen.

Antibody, monoclonal: A unique, isolated antibody molecule produced by ahybridoma.

Antibody, monospecific: A polyclonal antibody produced in immunologicalresponse to a single or few epitopes found in (1) a short, isolated,synthetic antigen or (2) a short, isolated, carrier-bound hapten.

Antigen: A molecule or composition of matter which (1) induces an immuneresponse in an animal, and (2) interacts specifically withantigen-recognizing components of an immune animal's immune system.

Asyntactic: Not having the same arrangement (syntax); “out of register.”In particular, note that fusion proteins cannot result from theasyntactic linkage of two (or more) open reading frames.

Carrier: A molecule required in combination with a hapten in order foran immune response to the hapten to occur. That is, a molecule whichputs a hapten in a molecular context in which the hapten has enhancedimmunogenicity.

Detectable label: A chemical moiety that is coupled to a biomolecule toenable detection of the biomolecule and which may be selected from thegroup consisting of a radiolabel, an enzyme such as horseradishperoxidase or alkaline phosphatase, streptavidin, biotin, an epitoperecognized by an antibody, and equivalents thereof.

Detectabty labeled: A state of a biomolecule in which the biomoleculehas covalently attached to it a detectable label.

Disease: (1) Excludes pregnancy per se but not autoimmune and otherdiseases associated with pregnancy; (2) includes any abnormal conditionof an organism or part, especially as a consequence of infection,inherent weakness, environmental stress, that impairs normalphysiological functioning; and (3) includes cancers and tumors.

DNA sequence: The sequence of contiguous nucleotide bases of a strand ofDNA as read from 5′ to 3′. Synonymous with “DNA molecule.”

Enzyme: Protein that is a catalyst for a specific chemical reaction,often one involving one or more biomolecules as substrates and/orproducts. Unlike non-biologically derived catalysts, enzymes mayrecognize a substrate with stereospecificity, i.e., some enzymes arecapable of recognizing, and thus catalyzing the chemical reaction of,only one of a pair of L- and D-enantiomers.

Epitope: A part of an antigen that interacts specifically withantigen-recognizing components of an animal's immune system. In apolypeptidic antigen, epitopes may correspond to short sequences ofcontiguous amino acids; the remainder of the antigen is called thecarrier. Synonymous with antigenic determinant.

Expression vector: An artificial DNA sequence, or a naturally-occurringDNA sequence that has been artificially modified, into which foreign orabnormal genes can be inserted and that contains transcription andtranslation signals that direct the expression of the inserted genes inhost cells appropriate for the expression vector, and the DNA of whichis replicated, either extra- or intra-chromosomally, in such appropriatehost cells.

Expression construct: A construct consisting essentially of anexpression vector and one or more foreign or abnormal genes insertedtherein in such a manner that the expression vector's transcription andtranslation signals are operably linked to the inserted gene(s).

Foreign or abnormal: Not endogenous to a healthy, wild-type organism.“Foreign or abnormal genes” designates nucleic acid sequences that arenot endogenous to an organism's genome, or originally endogenous nucleicacid sequences that have been rearranged, mutated, or otherwisegenetically engineered so as to possess properties (i.e., genomiclocation, regulation of expression, copy number, etc.) not possessed bythe endogenous nucleic acid sequences from which they were derived.

Gene: A DNA sequence that consists of a structural gene, e.g., a readingframe that encodes a polypeptide sequence, according to the standardgenetic code; and expression elements, e.g., promoters, terminators,enhancers, etc., required for transcription of the structural gene.

Genetically engineered: Subject to human manipulation intended tointroduce genetic change.

Hapten: A small molecule which (1) cannot, by itself, induce an immuneresponse in an animal, (2) can, in combination to a carrier to which itis bound, induce an immune response in an animal, and (3) interactsspecifically with the antigen-recognizing components of an immuneanimal's immune system.

Host animal: An animal that harbors foreign and/or abnormal genesintroduced as a result of (1) invasion of cells of the animal by anaturally occurring or genetically engineered intracellular parasite; or(2) introduction into cells of foreign or abnormal genes by humanmanipulation.

Immune animal: An animal which has been presented with an immunizingamount of antigen and has generated a humoral and/or cell-mediatedimmune response thereto.

Mammal: (1) Excludes human beings, individually and collectively, in allstages of development, including embryonic and fetal stages, unlessotherwise indicated; and (2) includes all other animals that are membersof the vertebrae class Mammalia, including an individual animal in anystage of development, including embryonic and fetal stages, whereinmembers of the class are distinguished by self-regulating bodytemperature, hair, and, in the females, milk-producing mammae.

Microorganism: A single-celled organism (e.g., a bacterium) or anintracellular parasite (e.g., a rickettsia or a virus); includes both“live” and “attenuated” microorganisms.

Operably linked: Arranged so as to have a functional relationship; inexpression constructs, inserted foreign or abnormal genes that areproperly positioned with regard to the signals that controltranscription and translation so that efficient expression of theinserted genes occurs are said to be operably linked to such signals(and vice-versa).

Potypeptide: A polymer of amino acid residues.

Protein: A biomolecule comprising one or more polypeptides arranged intoa functional, three-dimensional form.

Restriction endonuclease: An endonuclease that cleaves DNA at eachoccurrence therein of a specific recognition sequence. Synonymous with“restriction enzyme.”

Syntactic: Having the same arrangement (syntax); “in register.” Inparticular, note that fusion proteins can result from the syntacticlinkage of two (or more) open reading frames.

Transgene: A gene that does not occur naturally in an animal, i.e., aforeign or abnormal gene, introduced into an animal by nonnatural means,i.e., by human manipulation.

Transgenic animal: An animal into which has been introduced, bynonnatural means, i.e., by human manipulation, one or more transgenes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In its broadest form, the invention comprises a novel mammalian proteinknown as “ARF-p19,” that regulates the cell cycle; nucleic acidmolecules having sequences encoding polypeptide sequences of ARF-p19;antibodies specific for ARF-p19; transgenic non-human animals withalterations in the gene encoding ARF-p19; methods of making ARF-p19nucleic acids and polypeptides; methods of making ARF-p19-specificantibodies; methods of making transgenic non-human animals withalterations in the gene encoding ARF-p19; and methods of using thenucleic acids, proteins, antibodies and transgenic animals of theinvention to detect ARF-p19 nucleic acids or proteins in a sample, todiagnose cancers or predispositions thereto, to evaluate compositionsfor their therapeutic or oncogenic potential, and to prepare therapeuticcompositions for the treatment of tumors and cancers.

Nucleic Acids and Related Embodiments

In one embodiment, the invention comprises nucleic acids havingsequences encoding mouse ARF-p19, human ARF-p19 or ARF-p19 polypeptidesfrom other mammals. For example, the invention provides cDNA moleculesencoding mouse ARF-p19 (SEQ ID NO:1). The ARF-p19 cDNAs of the inventionare in turn used to isolate additional nucleic acids that encode ARF-p19polypeptide sequences, such as mouse and human genomic DNA clones.Moreover, because the homology between the nucleotide sequences of mouseand human ARF-p19 genes is quite high, the mouse and human nucleic acidsmay be used to design probes or degenerate primers for PCR in order toisolate cDNA and genomic clones of ARF-p19 genes from other mammals.

One skilled in the art can readily adapt the nucleic acid sequences ofthe invention to any system which is capable of producing nucleic acidsto produce the nucleic acids of the invention. The nucleic acids of theinvention, which may optionally comprise a detectable label, may beprepared as cDNA clones, genomic clones, RNA transcribed from eithercDNA or genomic clones, synthetic oligonucleotides, and/or syntheticamplification products resulting, e.g., from PCR. The nucleic acids ofthe invention may be prepared in either single- or double-stranded form.

Methods of preparing cDNA clones are known in the art (see, for example,Chapter 8 in Sambrook et al., Molecular Cloning: A Laboratory Manual,Vol. 2, 2d. Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989), pages 8.1-8.86). Methods of analyzing genomic DNAsequences and preparing genomic clones are known in the art (see, forexample, Chapter 9 in Sambrook et al., Molecular Cloning: A LaboratoryManual, Vol. 2, 2d. Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), pages 9.1-9.62; and Chapter 2 in CurrentProtocols in Molecular Biology, Vol. 1, Ausubel et al., eds., John Wiley& Sons, Inc., Boston, Mass. (1994), pages 2.1.1-2.14.8). Genonmic DNAsequences, i.e., chromosomally-derived nucleic acids, are isolated (seeExample 9) from mice and other non-human animals and used for theproduction of transgenic non-human animals. RNA containing ARF-p19sequences may be prepared from cells expressing ARF-p19 according tomethods known in the art (see, e.g., Chapter 4 in Current Protocols inMolecular Biology, Vol. 1, Ausubel et al., eds., John Wiley & Sons,Inc., Boston, Mass. (1994), pages 4.1.1-4.10.11), or may be generated byin vitro transcription using the DNA molecules of the invention (see,e.g., Chapter 10 in Sambrook et al., Molecular Cloning: A LaboratoryManual, Vol. 2, 2d. Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), pages 10.1-10.70).

Synthetic oligonucleotides having ARF-p19-specific nucleotide sequencescan be prepared using the nucleic acid sequences of the invention byknown methods (see, e.g., Chapter 11 in Sambrook et al., MolecularCloning: A Laboratory Manual, Vol. 2, 2d. Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), pages 11.1-11.61).When used as primers in the polymerase chain reaction (PCR), thesynthetic oligonucleotides preferably contain from about 15 to about 30contiguous nucleotides exactly corresponding to unique portions of theARF-p19 sequences of the invention, but may optionally containadditional nucleotides 5′ therefrom (Innis, M. A. and Gelfand, D. H.,Chapter 1 in PCR Protocols: A Guide to Methods and Applications, Inniset al., eds., Academic Press, Inc., New York (1990), pages 3-12; Saiki,R. K., Chapter 2 in PCR Protocols: A Guide to Methods and Applications,Innis et al., eds., Academic Press, Inc., New York (1990), pages 13-20).Synthetic amplification products are prepared using the syntheticoligonucleotides of the invention in amplification systems such as PCR(see, e.g., U.S. Pat. No. 4,965,188 to Mullis et al. (Oct. 23, 1990);Scharf, S. J., Chapter 11 in PCR Protocols: A Guide to Methods andApplications, Innis et al., eds., Academic Press, Inc., New York (1990),pages 84-98; Chapter 15 in Current Protocols in Molecular Biology, Vol.2, Ausubel et al., eds., John Wiley & Sons, Inc., Boston, Mass. (1994),pages 15.0.1-15.8.8; and Chapter 14 in Sambrook et al., MolecularCloning: A Laboratory Manual, Vol. 2, 2d. Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), pages 14.1-14.35).Those of skill in the art will appreciate that chemical derivatives ofnucleotide structures can be substituted for natural nucleotides in thenucleic acids of the invention.

Methods of nucleic acid expression: In one aspect of this embodiment ofthe invention, the nucleic acids of the invention are used to prepareARF-p19 proteins, or fusion proteins derived from ARF-p19, viarecombinant DNA technology. By inserting any of the nucleic acids of theinvention that encode ARF-p19 polypeptide sequences into an appropriateexpression vector, and introducing the resultant expression vectorconstruct into appropriate host cells, those skilled in the art canproduce large quantities of ARF-p19 polypeptides.

There are numerous host/expression vector systems available for thegeneration of proteins from the isolated nucleic acids of the invention.These include, but are not limited to, bacteria/plasmid systems,bacteria/phage systems, eukaryotic cell/plasmid systems, eukaryoticcell/virus systems, and the like (see, for example, U.S. Pat. No.4,440,859 to Rutter et al. (Apr. 3, 1984); Chapter 16 in CurrentProtocols in Molecular Biology, Vol. 2, Ausubel et al., eds., John Wiley& Sons, Inc., Boston, Mass. (1994), pages 16.0.5-16.20.16; and Sambrooket al., Molecular Cloning: A Laboratory Manual, Vol. 3, 2d. Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Oneskilled in the art can readily adapt the nucleic acids of the inventionto any host/expression vector system which is capable of propagating andexpressing heterologous nucleic acids to produce the proteins orpolypeptides of the invention. Preferred host/expression systems includebacteria/plasmid systems and insect cell/baculoviral expression vectorsystems.

Diagnostic methods and kits: In another aspect of this embodiment,ARF-p19 nucleic acid sequences are used to prepare oligonucleotideprobes, or PCR primers, to serve as materials for diagnostic tests forARF-p19 expression, mutation, or deletion in samples of cells isolatedfrom mammals. Deletions of the genes encoding p15 and p16 occurfrequently in cancer cells, and the resulting loss of theiranti-proliferative functions can contribute to tumorigenesis (Noburi etal., Nature 368:753-756 (1994)). Similarly, point mutations, deletionsor other mutations in the genes encoding ARF-p19 are diagnostic ofcancer or indicative of a predisposition to develop certain types ofcancers.

Mutations in the human gene for ARF-p19 are detected by any of a varietyof methods depending in part on the nature of the mutation of interest.Deletions and insertions of about 100 base pairs (bp) or more aredetected by electrophoretic separation of genomic DNA and hybridizationanalysis using nucleic acid probes derived from unique portions of thenucleotide sequence of the human ARF-p19 coding sequence (SEQ ID NO:3;see also FIG. 7), or by PCR of genomic DNA using syntheticoligonucleotides derived from the unique portions of the nucleotidesequence of the human ARF-p19 coding sequence as primers. The term “theunique portions of the nucleotide sequence of human ARF-p19” is intendedto encompass nucleotide sequences that occur in molecules encodingARF-p19 but which are not found in p16-InK4a mRNAs.

In one aspect, the invention comprises methods of detecting the presenceof a nucleic acid polymorphism associated with a predisposition todevelop cancer by analyzing DNA or RNA from a mammal using nucleic acidmolecules containing part or all of the unique portions of thenucleotide sequences from an ARF-p19 gene from a mammal, such as a mouseor a human, or the reverse complement thereof. Such methods are used inconjunction with any procedure which will detect the nucleic acids ofthe invention. Examples of such procedures include hybridizationanalysis using the nucleic acids of the invention, i.e., isolation ofnucleic acids from the cells of a mammal, followed by restrictiondigestion, separation by a means such as gel electrophoresis, transferto nitrocellulose or a comparable material, and detection of ARF-p19nucleic acid sequences thereon by exposure to detectably labeled nucleicacid probes which contain nucleotide sequences encoding ARF-hp19polypeptide sequences.

In one embodiment of the present invention, the preferred method ofdetecting the presence of a DNA polymorphism associated with apredisposition to develop cancer involves RFLP (restriction-fragmentlength polymorphism) techniques based on amplification of ARF-p19sequences via PCR, followed by restriction digestion and agarose gelelectrophoresis. In this method, a biological sample containingnucleated cells, preferably leukocytes, is obtained from a human.Suitable biological samples having nucleated cells that may be used inthis invention include, but are not limited to, blood and tissue. Themethod of obtaining the biological sample will vary depending upon thenature of the sample. By the term “nucleated cells” is meant any cellcontaining a nucleus. Examples of such cells include, but are notlimited to, white blood cells, epithelial cells, or mesenchymal cells.The cells are then isolated from the sample and the DNA from thenucleated cells is purified using conventional methods known in the artsuch as phenol-chloroform extraction, lytic enzymes, chemical solutionsand centrifugation, or size exclusion chromatography (see, for example,Blin and Stafford, Nucl. Acid Res. 3:2303-2308 (1976); Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982)). Following isolation, the DNA sequencesof interest are amplified using conventional PCR methods (see, forexample, Innis et at., PCR Protocols, Academic Press, N.Y. (1990);Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986);Mullis and Faloona, Methods Enzymol. 155:335-350 (1987); and Mullis etal., U.S. Pat. No. 4,965,188 (Oct. 23, 1990)).

In one aspect of this embodiment, sequences comprising the uniqueportions of nucleotide sequences for ARF-p19 are utilized as primers forspecific amplification of ARF-p19 nucleic acids (see Example 2). In suchan embodiment the amplified product is subjected to restrictiondigestion prior to visualization. Different alleles of ARF-INK4a willyield amplified fragments of differing size after digestion with anappropriate restriction endonuclease.

The amplified DNA is then precipitated, and digested with a restrictionenzyme, such as BamHI, BgII, PstI, or EcoRI. Digested DNA fragments areseparated according to their molecular weights to form a pattern,typically using agarose gel electrophoresis. Following electrophoresis,the gel is stained with an appropriate agent, such as ethidium bromide,using standard protocols, and photographed under ultraviolettransillumination. Polymorphisms result in the appearance of additionalbands (i.e., bands not found in the wild-type ARF-InK4a allele) on thegel.

In an alternative aspect of this embodiment, the DNA isolated from thecells' nuclei is digested with a given restriction endonuclease,utilizing PCR amplification. The restriction endonucleases that may beused in this invention include, but are not limited to, BamHI, BgM,PstI, or EcoRi. After a digest is obtained, and the DNA is separated bystandard technique, for example by agarose gel electrophoresis, theseparated bands are probed with one or more DNA fragments containing aunique portion of the nucleotide sequences encoding human ARF-p19polypeptide sequences. In one aspect of this embodiment, the preferredprobe of the invention is based on the cDNA or genomic sequence from thegene for human ARF-p19.

The use of RFLP technology is only one preferred embodiment of detectingpolymorphisms in the nucleic acids of the invention. Since, ultimately,the use of RFLP depends on polymorphism in DNA restriction sites alongthe nucleic acid molecule, other methods of detecting the polymorphismcan also be used. Any method of analysis which allows one skilled in theart to determine the linkage between the polymorphism detected by theprobes and primers of the present invention can be utilized. Techniquessuch as direct location of a polymorphism affecting ARF-p19 at itschromosomal location by in situ hybridization (e.g., FISH) usingradiolabeled, fluorescence-labeled, or enzyme-labeled probes may beemployed. Other suitable techniques include, but are not limited to,amplification methods such as the ribonuclease mis-match cleavage assayand direct oligonucleotide hybridization.

Any size fragment of the human InK4A gene (SEQ ID NO:3) can be utilizedas a probe as long as it is capable of hybridizing to a restrictionfragment which displays a polymorphism within an intron or an exonrequired for ARF-p19 expression. The hybridization probes can be labeledby standard labeling techniques such as with a radiolabel, enzyme label,fluorescent label, biotin-avidin label, chemiluminescence, and the like.After hybridization, the probes are visualized using known methods.Comparison of the RFLP or RFLP's for the subject under investigationwill quickly reveal the presence or absence of polymorphisms in the geneencoding human ARF-p19 linked to a predisposition to cancer.Polymorphisms that may be detected by the methods of the inventioninclude RFLPs, point mutations, insertions, deletions, inversions,alternately spliced mRNAs, and the like.

The materials for use in this aspect of the invention are ideally suitedfor the preparation of a kit. Specifically, the invention provides acompartmentalized kit to receive in close confinement, one or morecontainers which comprises: (a) a first container comprising one or moreof the probes or amplification primers of the present invention; and (b)one or more other containers comprising one or more of the following: asample reservoir, wash reagents, reagents capable of detecting presenceof bound probe from the first container, or reagents capable ofamplifying sequences hybridizing to the amplification primers.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers or strips of plastic or paper. Suchcontainers allows one to efficiently transfer reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated and the agents or solutions of each containercan be added in a quantitative fashion from one compartment to another.Such containers will include a container which will accept the testsample, a container which contains the probe or primers used in theassay, containers which contain wash reagents (Tris-buffers, etc.), andcontainers which contain the reagents used to detect the bound probe oramplified product.

Types of detection reagents include labeled secondary probes, or in thealternative, if the primary probe is labeled, the enzymatic, or antibodybinding reagents which are capable of reacting with the labeled probe.One skilled in the art will readily recognize that the disclosed probesand amplification primers of the present invention can readily beincorporated into one of the established kit formats which are wellknown in the art. In one example, a first container may contain ahybridization probe. The second container may contain the restrictionenzyme to be used in the digest. Other containers may contain reagentsuseful in the localization of labeled probes, such as enzyme substratessuch as x-gal tagged avidin if a biotinylated probe is utilized. Stillother containers may contain buffers, etc.

Gene therapy: In another embodiment of this invention, ARF-p19 nucleicacid sequences are used for gene therapy, i.e., to inhibit, enhance orrestore expression of ARF-p19 in cells with reduced, altered or noARF-p19 activity, using the nucleic acid sequences of the invention.

1. In order to enhance or restore ARF-p19 activity to cells in need ofgrowth regulation, ARF-p19 expression constructs are prepared. Anexpression construct consists of nucleic acid sequences encoding aprotein having ARF-p19 polypeptide sequences operably linked to nucleicacid sequences required for genetic expression in a cell (such aspromoters) in an expression vector. The expression constructs areintroduced into cells, wherein they direct expression of proteins havingARF-p19 polypeptide sequences. The expressed proteins may be fusionproteins that additionally include polypeptide sequences designed toimprove the in vivo activity, targeting and/or stability of the geneproducts expressed by the expression construct.

The expressed proteins function to restore or enhance ARF-p19 functionin their host cells and thus negatively regulate the progression of thecell through the cell cycle. The disclosure demonstrates that, even incells genetically engineered to overexpress cyclin D and thus possessing5-10 fold greater levels of CDKs than corresponding wild-type cells, theconstitutive expression of ARF-p19 in a cell results in G1 or G2 phasearrest (see Example 5). Thus, even in cells with runaway cyclin Dexpression, the introduction of ARF-p19 function in excess inhibits theprogression of the cells through the cell cycle and thus prevents theirfurther growth.

2. In order to inhibit ARF-p19 activity in cells in need of growthstimulation, synthetic antisense oligonucleotides are prepared from thecoding sequences for ARF-p19 found in cDNA clones. An antisenseoligonucleotide consists of nucleic acid sequences corresponding to thereverse complements of ARF-p19 coding sequences or other sequencesrequired to be present in ARF-p19 mRNA molecules for in vivo expression.The antisense oligonucleotides are introduced into cells, wherein theyspecifically bind to ARF-p19 MRNA molecules (and thus inhibittranslation of ARF-p19 gene products), or to double-stranded DNAmolecules to form triplexes (see U.S. Pat. No. 5,190,931 to Inouye (Mar.2, 1993); Riordan and Martin, Nature 350:442443 (1991)).

Because antisense oligonucleotides bind with high specificity to theirtargets, selectivity is high and toxic side effects resulting frommisdirection of the compounds are minimal, particularly given thepresent state of the art with regard to the design of, preparation andchemical modification of, and means of delivery to cells for,oligonucleotides (see, e.g., Wagner, R. W., Nature 372:333-335 (1994);Tseng and Brown, Cancer Gene Therapy 1:65-71 (1994); Morishita, R., etal., J. Clin. Invest. 93:1458-1464 (1994); Stein and Cheng, Science261:1004-1012 (1993); Lisziewicz, J., et al., Proc. Natl. Acad. Sci.(USA) 90:3860-3864 (1993); Watson, P. H., et al., Cancer Res.51:3996-4000 (1991); Han, L., et al., Proc. Natl. Acad. Sci. (USA)88:4313-4317 (1991); Florini and Ewton, J. Biol. Chem. 265:13435-13437(1990); and Ullmarm and Peyman, Chem. Reviews 90:543-583 (1990)). Meansfor the delivery of oligonucleotides to cells include, but are notlimited to, liposomes (see, e.g., Renneisen, K., et al., J. Biol. Chem.265:16337-16342 (1990)) and introduction of expression constructs thatdirect the transcription of antisense oligoribonucleotides in vivo (see,e.g., Shohat, O., et al., Oncogene 1:277-283 (1987)).

Polypeptides and Related Embodiments

In one embodiment, the invention comprises proteins having amino acidsequences of mouse ARF-p19 protein, human ARF-p19 protein and ARF-p19polypeptides from other mammals. For example, the invention provides thecomplete amino acid sequences of mouse ARF-p19 (SEQ ID NO:2) and ofhuman ARF-p19 (SEQ ID NO:4). When introduced into mammalian cellsARF-p19 proteins induce cell cycle arrest or, at lower concentrations,slow cell growth to a desired rate.

One skilled in the art can readily adapt the amino acid sequences of theinvention to a variety of known applications. For example, fusionproteins that comprise amino acid sequences from ARF-p19 and a secondpolypeptide can be produced by recombinant DNA technology to generatenovel proteins having properties of both parent proteins (see Example4). Similarly, the proteins of the invention can be conjugated to otherproteins in order to target the conjugated protein to CDK-cyclincomplexes in a cell. Synthetic oligopeptides (a.k.a. “peptides”)generally contain from about 5 to about 100 contiguous amino acidsexactly corresponding to the polypeptide sequence of ARF-p19 of theinvention, but may optionally contain additional amino acids at thecarboxyl terminus, the amino terminus, or both. Moreover, those of skillin the art will appreciate that substitution of endogenous amino acidsfor chemical derivatives and/or isomers of amino acids will yieldpeptides with properties that are enhanced relative to the nativeARF-p19 proteins. Properties that may be so altered include, forexample, in vivo stability.

Antibodies and Related Embodiments

In another embodiment of the invention, ARF-p19 proteins, oroligopeptide sequences derived therefrom, are used to create antibodycompositions that specifically recognize (bind) ARF-p19 epitopes.Antibodies to ARF-p19 serve as probes for diagnostic tests for ARF-p19expression or as diagnostic materials. Antibodies to ARF-p19 can also beconjugated to toxins to generate specific immunotoxins for use inmammalian therapy.

Methods of generating antibodies using purified proteins or syntheticoligopeptides are known in the art (see Antibodies: A Laboratory Manual,Harlow, E., and Lane, D., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1988)). The antibody compositions of the invention may bepolyclonal, monospecific or monoclonal.

Diagnostic methods and kits: In one aspect of this embodiment, theconcentration of ARF-p19 protein in a sample of cells from a mammal isdetermined by contacting the sample with a detectably labeled antibodycomposition specific to ARF-p19, qualitatively or quantitativelydetermining the amount of label bound or not bound in the sample, andcalculating therefrom the concentration of ARF-p19 in the sample. Thesample of cells is obtained from a mammal and are washed in anappropriate buffer such as Hank's balanced salt solution. The cells arelysed and incubated with a detectably labeled ARF-p19-specific antibodycomposition for an appropriate amount of time. The cells are washed withthe buffer a second time to remove unbound antibody. The amount of boundor unbound labeled antibody is then detected by conventional means.

Alternatively, unlabeled ARF-p19-specific antibody compositions, boundor unbound in a sample, are detected using a secondary antibody orprotein which is specific for an immunoglobulin, e.g., protein A,protein G, anti-IgM or anti-IgG antibodies. In this alternativeembodiment, the secondary (anti-immunoglobulin) antibodies, which may bemonoclonal or polyclonal, are detectably labeled and are detected in thecourse of carrying out the method.

Alternatively, ARF-p19 levels in a sample of mammalian cells aredetermined by detecting the level of soluble ARF-p19 in a sample oflysed cells. In this aspect, a sample of lysed cells obtained from amammal is contacted with an ARF-p19-specific antibody composition whichis immobilized onto a solid matrix, and allowed to incubate so as toform an ARF-p19/ARF-p19-specific antibody complex. Following a wash stepwith suitable buffers to remove the unbound antibody, a detectablylabeled molecule which binds to the ARF-p19-specific antibodycomposition is added. The amount of bound label then is detected todetermine the concentration of ARF-p19 present in the sample. Suitabletypes of immunoassays for detecting ARF-p19 include sandwich immunoassayand competition assays, performed using conventional methods. Naturally,other ligands specific for ARF-p19 may be used in lieu ofARF-p19-specific antibody compositions.

Of course, the specific amounts of ARF-p19-specific antibodycompositions and detectably labeled second antibodies, the temperatureand time of incubation, as well as other assay conditions may be varied,depending on various factors including the concentration of ARF-p19 inthe sample, the nature of the sample, and the like. Those skilled in theart will be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation. Other suchsteps as washing, stirring, shaking, filtering and the like may be addedto the assays as is customary or necessary for the particular situation.

A variety of means may be used to detectably label antibody compositionsfor use in the methods of the invention. For example, one means by whichan ARF-p19-specific antibody composition, or secondary antibodies, canbe detectably labeled is by conjugation to an enzyme. The conjugatedenzyme, when later exposed to its substrate, will react with thesubstrate in such a manner as to produce a chemical moiety which can bedetected, for example, by spectrophotometric, fluorometric or by visualmeans. Enzymes which can be used to detectably label antibodycompositions include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-v-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholineesterase. Antibody compositions may also be labeled with a radioactiveisotope which can be determined by such means as the use of a gammacounter or a scintillation counter or by autoradiography. It is alsopossible to label antibody compositions with a fluorescent compound.When fluorescently labeled antibody is exposed to light of the properwave length, its presence can then be detected due to the fluorescenceof the dye. Among the most commonly used fluorescent labeling compoundsare fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde and fluorescamine. Antibodies can alsobe detectably labeled using fluorescence emitting metals such as ¹⁵²Eu,or others of the lanthanide series. These metals can be attached toARF-p19-specific antibodies using such metal chelating groups asdiethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA). Antibodies also can be detectably labeled by coupling to achemiluminescent compound. The presence of chemiluminescent-taggedantibodies is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridium ester, imidazole, acridinium salt andoxalate ester. Likewise, a bioluminescent compound may be used to labelantibody compositions for use in the methods of the present invention.Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent protein isdetermined by detecting the presence of luminescence. Importantbioluminescent compounds for purposes of labeling are luciferin,luciferase and aequorin.

Detection of bound or unbound antibodies may be accomplished by ascintillation counter, for example, if the detectable label is aradioactive gamma emitter, or by a fluorometer, for example, if thelabel is a fluorescent material. In the case of an enzyme label, thedetection can be accomplished by colorimetric methods which employ asubstrate for the enzyme. Detection may also be accomplished by visualcomparison of the extent of enzymatic reaction of a substrate incomparison with similarly prepared standards.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the previously describedimmunoassays with ARF-p19-specific antibodies, in order to diagnosecertain types of cancers, or to detect a predisposition for certaintypes of cancers, in a mammal.

Specifically, the invention provides a compartmentalized kit to receive,in close confinement, one or more containers which comprises: (a) afirst container containing an ARF-p19-specific antibody; and (b) one ormore other containers containing one or more of the following: washreagents, and reagents capable of detecting presence of bound or unboundARF-p19-specific antibodies.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers or strips of plastic or paper. Suchcontainers allow one to efficiently transfer reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother. Such containers will include a container which will accept thetest sample, a container which contains the antibodies used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, etc.), and containers which contain thereagents used to detect the bound antibody.

Types of detection reagents include detectably labeled secondaryantibodies, or in the alternative, if the primary antibody is detectablylabeled, the appropriate enzymatic or antibody binding reagents whichare capable of reacting with the labeled antibody. One skilled in theart will readily recognize that the disclosed antibodies of the presentinvention can readily be incorporated into any one of the variety ofestablished kit formats which are well known in the art.

Therapeutics and Related Embodiments

Another embodiment of the invention includes screening for and producingnew compounds that inhibit the activity of ARF-p19, to be applied tomammalian cells in need of reduced regulation of their cell cycles,cellular growth, and/or DNA replication. For example, in order topromote cellular growth in, e.g., healing processes, “negative-dominant”(Herskowitz, I., Nature 329:219-222 (1987)) ARF-p19 variants areprepared which competitively inhibit endogenous ARF-p19 proteins andthereby reduce ARF-p19 activity within a cell. As another example of ameans by which cellular growth may be promoted, antibodies that bindregions of ARF-p19 involved in its biological action are introduced intoa cell and prevent endogenous ARF-p19 proteins from functioning, therebyreducing ARF-p19 activity within a cell.

In a related embodiment, proteins, fusion proteins, conjugates, orsynthetic oligopeptides having ARF-p19 function can be introduced intoeukaryotic cells to arrest their progression from G1 to S phases, orfrom G2 to M phases, during interphase and thus inhibit growth ofundesired cells, e.g., cancer cells (see Example 5). ARF-p19, orderivatives thereof, can be employed in combination with conventionalexcipients, i.e., pharmaceutically acceptable organic or inorganiccarrier substances suitable for parenteral application which do notdeleteriously react with the active compound. Suitable pharmaceuticallyacceptable carriers include, but are not limited to, water, saltsolutions, alcohol, vegetable oils, polyethylene glycols, gelatin,lactose, amylose, magnesium stearate, talc, silicic acid, viscousparaffin, perfume oil, fatty acid monoglycerides and diglycerides,petroethral fatty acid esters, hydroxymethylcellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringand/or aromatic substances and the like which do not deleteriously reactwith the active compounds. For parenteral application, particularlysuitable vehicles consist of solutions preferably oily or aqueoussolutions, as well as suspensions, emulsions, or implants. Aqueoussuspensions may contain substances which increase the viscosity of thesuspension and include, for example, sodium carboxymethyl cellulose,sorbitol, and/or dextran. Optionally, the suspension may also containstabilizers.

The term “therapeutically effective amount,” for the purposes of theinvention, refers to the amount of ARF-p19 or derivatives thereof whichis effective to achieve its intended purpose. While individual needsvary, determination of optimal ranges for effective amounts of ARF-p19or its derivatives is within the skill of the art. Generally, the dosagerequired to provide an effective amount of the composition, and whichcan be adjusted by one of ordinary skill in the art will vary, dependingon the age, health, physical condition, weight, extent of disease of therecipient, frequency of treatment and the nature and scope of thedesired effect.

Transgenic Animals and Related Embodiments

In another embodiment of the invention, ARF-p19 nucleic acid sequencescan be used to create transgenic non-human animals to serve as animalmodels for ARF-p19 overexpression (transgenic expression) or mutationssuch as multiple stop codons (“knockouts” or “null alleles”) or othermutations which alter one or more ARF-p19 activities without affectingInK4a-p16 activity. For example, transgenic mice having little or noARF-p19 activity due to mutations in one or both alleles of the geneencoding ARF-p19 (INK4A) are prone to develop certain types of tumors.

The non-human animals of the invention comprise any animal having adeficiency of ARF-p19 activity as a result of the transgenic alterationof the gene(s) encoding ARF-p19. Such non-human animals includevertebrates such as rodents, non-human primates, sheep, dog, cow,amphibians, reptiles, etc. Preferred non-human animals are selected fromnon-human mammalian species of animals, most preferably, animals fromthe rodent family including rats and mice, most preferably mice.

The transgenic animals of the invention are animals into which has beenintroduced by nonnatural means (i.e., by human manipulation), one ormore genes that do not occur naturally in the animal, e.g., foreigngenes, genetically engineered endogenous genes, etc. The nonnaturallyintroduced genes, known as transgenes, may be from the same or adifferent species as the animal but not naturally found in the animal inthe configuration and/or at the chromosomal locus conferred by thetransgene. Transgenes may comprise foreign DNA sequences, i.e.,sequences not normally found in the genome of the host animal.Alternatively or additionally, transgenes may comprise endogenous DNAsequences that are abnormal in that they have been rearranged or mutatedin vitro in order to alter the normal in vivo pattern of expression ofthe gene, or to alter or eliminate the biological activity of anendogenous gene product encoded by the gene. (Watson, J. D., et al., inRecombinant DNA, 2d Ed., W. H. Freeman & Co., New York (1992), pages255-272; Gordon, J. W., Intl. Rev. Cytol. 115:171-229 (1989); Jaenisch,R., Science 240:1468-1474 (1989); Rossant, J., Neuron 2:323-334 (1990)).

Methods of preparing transgenic animals: In one aspect of thisembodiment of the invention, the nucleic acids of the invention are usedto prepare transgenic constructs to be introduced into non-human animalsin order to generate the transgenic animals of the invention.Specifically, ARF-p19 sequences derived from the genome of the non-humananimal of choice are used to create such transgenic constructs.

The transgenic non-human animals of the invention are produced byintroducing ARF-p19 transgenic constructs into the germline of thenon-human animal. Embryonic target cells at various developmental stagesare used to introduce the transgenes of the invention. Different methodsare used depending on the stage of development of the embryonic targetcell(s).

1. Microinjection of zygotes is the preferred method for incorporatingtransgenes into animal genomes in the course of practicing theinvention. A zygote, a fertilized ovum that has not undergone pronucleifusion or subsequent cell division, is the preferred target cell formicroinjection of transgenic DNA sequences. The murine male pronucleusreaches a size of approximately 20 micrometers in diameter, a featurewhich allows for the reproducible injection of 1-2 picoliters of asolution containing transgenic DNA sequences. The use of a zygote forintroduction of transgenes has the advantage that, in most cases, theinjected transgenic DNA sequences will be incorporated into the hostanimal's genome before the first cell division (Brinster, et al., Proc.Natl. Acad. Sci. (USA) 82:4438-442 (1985)). As a consequence, all cellsof the resultant transgenic animals (founder animals) stably carry anincorporated transgene at a particular genetic locus, referred to as atransgenic allele. The transgenic allele demonstrates Mendelianinheritance: half of the offspring resulting from the cross of atransgenic animal with a non-transgenic animal will inherit thetransgenic allele, in accordance with Mendel's rules of randomassortment.

2. Viral integration can also be used to introduce the transgenes of theinvention into an animal. The developing embryos are cultured in vitroto the developmental stage known as a blastocyte. At this time, theblastomeres may be infected with appropriate retroviruses (Jaenich, R.,Proc. Natl. Sci. (USA) 73:1260-1264). Infection of the blastomeres isenhanced by enzymatic removal of the zona pellucida (Hogan, et al., inManipulating the Mouse Embryo, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1986)). Transgenes are introduced via viral vectors whichare typically replication-defective but which remain competent forintegration of viral-associated DNA sequences, including transgenic DNAsequences linked to such viral sequences, into the host animal's genome(Jahner, et al., Proc. Natl. Acad. Sci. (USA) 82:6927-6931 (1985); Vander Putten, et al., Proc. Natl. Acad. Sci. (USA) 82:6148-6152 (1985)).Transfection is easily and efficiently obtained by culture ofblastomeres on a mono-layer of cells producing the transgene-containingviral vector (Van der Putten, et al., Proc. Natl. Acad. Sci. (USA)82:6148-6152 (1985); Stewart, et al., EMBO Journal 6:383-388 (1987)).Alternatively, infection may be performed at a later stage, such as ablastocoele (Jahner, D., et al., Nature 298:623-628 (1982)). In anyevent, most transgenic founder animals produced by viral integrationwill be mosaics for the transgenic allele; that is, the transgene isincorporated into only a subset of all the cells that form thetransgenic founder animal. Moreover, multiple viral integration eventsmay occur in a single founder animal, generating multiple transgenicalleles which will segregate in future generations of offspring.Introduction of transgenes into germline cells by this method ispossible but probably occurs at a low frequency (Jahner, D., et al.,Nature 298:623-628 (1982)). However, once a transgene has beenintroduced into germline cells by this method, offspring may be producedin which the transgenic allele is present in all of the animal's cells,i.e., in both somatic and germline cells.

3. Embryonic stem (ES) cells can also serve as target cells forintroduction of the transgenes of the invention into animals. ES cellsare obtained from pre-implantation embryos that are cultured in vitro(Evans, M. J., et al., Nature 292:154-156 (1981); Bradley, M. O., etal., Nature 309:255-258 (1984); Gossler, et al., Proc. Natl. Acad. Sci.(USA) 83:9065-9069 (1986); Robertson et al., Nature 322:445-448 (1986);Robertson, E. J., in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, Robertson, E. J., ed., IRL Press, Oxford (1987),pages 71-112). ES cells, which are commercially available (from, e.g.,Genome Systems, Inc., St. Louis, Mo.), can be transformed with one ormore transgenes by established methods (Lovell-Badge, R. H., inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach,Robertson, E. J., ed., IRL Press, Oxford (1987), pages 153-182).Transformed ES cells can be combined with an animal blastocyst,whereafter the ES cells colonize the embryo and contribute to thegermline of the resulting animal, which is a chimera (composed of cellsderived from two or more animals) (Jaenisch, R., Science 240:1468-1474(1988); Bradley, A., in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, Robertson, E. J., ed., IRL Press, Oxford (1987),pages 113-151). Again, once a transgene has been introduced intogermline cells by this method, offspring may be produced in which thetransgenic allele is present in all of the animal's cells, i.e., in bothsomatic and germline cells.

However it occurs, the initial introduction of a transgene is aLamarckian (non-Mendelian) event. However, the transgenes of theinvention may be stably integrated into germ line cells and transmittedto offspring of the transgenic animal as Mendelian loci. Othertransgenic techniques result in mosaic transgenic animals, in which somecells carry the transgenes and other cells do not. In mosaic transgenicanimals in which germ line cells do not carry the transgenes,transmission of the transgenes to offspring does not occur.Nevertheless, mosaic transgenic animals are capable of demonstratingphenotypes associated with the transgenes.

Transgenes may be introduced into animals in order to provide animalmodels for human diseases. Transgenes that result in such animal modelsinclude, e.g., transgenes that encode mutant gene products associatedwith an inborn error of metabolism in a human genetic disease andtransgenes that encode a human factor required to confer susceptibilityto a human pathogen (i.e., a bacterium, virus, or other pathogenicmicroorganism) (Leder et al., U.S. Pat. No. 5,175,383 (Dec. 29, 1992);Kindt et al., U.S. Pat. No. 5,183,949 (Feb. 2, 1993); Small et al., Cell46:13-18 (1986); Hooper et al., Nature 326:292-295 (1987); Stacey etal., Nature 332:131-136 (1988); Windle et al., Nature 343:665-669(1990); Katz et al., Cell 74:1089-1100 (1993)). Transgenic animals thatare predisposed to a disease may be used to identify compositions thatinduce the disease and to evaluate the pathogenic potential ofcompositions known or suspected to induce the disease (Berns, A. J. M.,U.S. Pat. No. 5,174,986 (Dec. 29, 1992)).

Offspring that have inherited the transgenes of the invention aredistinguished from littermates that have not inherited transgenes byanalysis of genetic material from the offspring for the presence ofbiomolecules that comprise unique sequences corresponding to sequencesof, or encoded by, the transgenes of the invention. For example,biological fluids that contain polypeptides uniquely encoded by thetransgenes of the invention may be immunoassayed for the presence of thepolypeptides. A more simple and reliable means of identifying transgenicoffspring comprises obtaining a tissue sample from an extremity of ananimal, e.g., a tail, and analyzing the sample for the presence ofnucleic acid sequences corresponding to the DNA sequence of a uniqueportion or portions of the transgenes of the invention. The presence ofsuch nucleic acid sequences may be determined by, e.g., hybridization(“Southern”) analysis with DNA sequences corresponding to uniqueportions of the transgene, analysis of the products of PCR reactionsusing DNA sequences in a sample as substrates and oligonucleotidesderived from the transgene's DNA sequence, etc.

Null alleles: A preferred embodiment is a transgenic animal that ishomozygous for a null (a.k.a. “knock-out”) allele of ARF-INK4A but whichhas a wild-type INK4A-p16 allele. For example, selective interruption ofINK4A exon 1β eliminates ARF-INK4A expression but does not affectsequences encoding InK4a-p16. Additionally or alternatively, one or morepoint mutations that create stop codons in the ARF-p19 reading frame,but which result in silent mutations in the InK4a-p16 reading frame, areintroduced by site-directed mutagenesis into cloned INK4A genomicnucleic acid sequences which are then reintroduced into the genome of ananimal to generate a transgenic ARF-p19-deficient animal. The transgenicARF-p19 null or ARF-p19-deficient animals of the invention arepredisposed to develop certain types of cancers, including but notlimited to melanomas, in a reproducible and thus reliable manner.

In order to generate null alleles in embryonic stem cells, thepositive-negative selection strategy of Mansour et al. (Nature336:348-352 (1988)) is applied. A positive selectable marker, forexample the hygromycin phosphotransferase cassette (van Deursen andWieringa, Nucl. Acids Res. 29:3815-3820 (1992)), is inserted into a 5′portion of an INK4 gene. This position for the positive selectablemarker is chosen to obtain a genuine null mutant allele, i.e., to avoidtranslation of a truncated polypeptide. In the resulting targetingvector the hygromycin gene is flanked 5′ and 3′ by several kb ofhomologous murine genomic sequences. In addition, a negative selectablemarker, for example the Herpes Simplex Virus (HSV) thymidine kinase (PK)gene, is placed in a 3′ position flanking the region of homologoussequences in order to enable selection against nonhomologous integrants.Both the positive and negative selectable markers are inserted in theantisense orientation with respect to the transcriptional orientation ofthe Ink4 gene, and are expressed due to the TK promoter and Py F441Polyoma enhancer. Linearized targeting construct is introduced into EScells by electroporation or other suitable means and selection withhygromycin and FIAU (1-[2-deoxy, 2-fluoro-β-D-arabinofuranosyl]) iscarried out for 7 to 10 days. Resistant colonies are expanded in 24-wellplates; half of the cells in each well are cryo-preserved and the otherhalf expanded for genotype analysis. Positive clones are stored inliquid nitrogen and thawed at least 3 days prior to blastocystinjection. Blastocysts are isolated, for example, at day 3.5 postcoitumby flushing the uterine horns of naturally mated C57BL/6 pregnantfemales with DMEM+10% FBS. Approximately 10 to 15 ES cells from eachhomologous recombinant clone with a normal karyotype are microinjectedinto recipient blastocysts, and about 10 to 20 embryos are transferredinto the uterine horns of (C57BL/6×CBA/Ca) F1 pseudopregnant fosters(Bradley, A., in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, Robertson, E. J., ed., IRL Press, Oxford (1987), pages113-151). Chimeric males are mated with C57BL/6 or FVB/J females andgermline transmission of the mutant allele is verified by Southern blotanalysis of tail DNA from F1 offspring with either agouti or gray coatcolor. F2 offspring from interbred heterozygotes are genotyped bySouthern blotting to identify homozygous null mutants.

Methods of evaluating the therapeutic or oncogenic potential ofcompositions: Using the transgenic animals of the invention, it ispossible to evaluate a variety of compositions for their therapeutic oroncogenic potential.

1. Generally, methods for determining the therapeutic potential of acomposition to treat cancer comprise the step of administering a knowndose of the composition to a transgenic animal having a phenotype ofreduced or altered ARF-p19 activity, monitoring resulting biological orbiochemical parameters correlated with cancer, and comparing thesymptoms of treated animals to those of untreated animals.

A first method of assessing the therapeutic potential of a compositionusing the transgenic animals of the invention comprises the steps of:

(1) Administering a known dose of the composition to a first transgenicanimal having a phenotype of reduced or altered ARF-p19 activity;

(2) Detecting the time of onset of cancer in the first transgenicanimal; and

(3) Comparing the time of onset of cancer in the first transgenic animalto the time of onset of cancer in a second transgenic animal having aphenotype of reduced or altered ARF-p19 activity, which has not beenexposed to the composition,

wherein a statistically significant decrease in the time of onset ofcancer in the first transgenic animal relative to the time of onset ofthe symptoms in the second transgenic animal indicates the therapeuticpotential of the composition for treating cancer.

A second method of assessing the therapeutic potential of a compositionusing the transgenic animals of the invention comprises the steps of:

(1) Administering a known dose of the composition to a first transgenicanimal having a phenotype of reduced or altered ARF-p19 activity, at aninitial time, t₀;

(2) Determining the extent of cancer in the first transgenic animal at alater time, t₁; and

(3) Comparing, at t₁, the extent of cancer in the first transgenicanimal to the extent of neurological symptoms in a second transgenicanimal having a phenotype of reduced or altered ARF-p19 activity, whichhas not been exposed to the composition at t₀,

wherein a statistically significant decrease in the extent of cancer att₁ in the first transgenic animal relative to the extent of the symptomsat t₁ in the second transgenic animal indicates the therapeuticpotential of the composition for treating cancer.

A third method of assessing the therapeutic potential of a compositionusing the transgenic animals of the invention comprises the steps of:

(1) Administering a known dose of the composition to a first transgenicanimal having a phenotype of reduced or altered ARF-p19 activity;

(2) Measuring the lifespan of the first trarsgenic animal; and

(3) Comparing the lifespan of the first transgenic animal to thelifespan of a second transgenic animal having a phenotype of reduced oraltered ARF-p19 activity, which has not been exposed to the composition,

wherein a statistically significant increase in the lifespan of thefirst transgenic animal relative to the lifespan of the secondtransgenic animal indicates the therapeutic potential of the compositionfor treating cancer.

2. Generally, methods for determining the potential of a composition tocause or exacerbate cancer comprise the step of administering a knowndose of the composition to a transgenic animals having a phenotype ofreduced or altered ARF-p19 activity, monitoring resulting biological orbiochemical parameters correlated with cancer, and comparing thesymptoms of treated animals to those of untreated animals.

A first method of assessing the oncogenic potential of a compositionusing the transgenic animals of the invention comprises the steps of:

(1) Administering a known dose of the composition to a first transgenicanimal having a phenotype of reduced or altered ARF-p19 activity;

(2) Detecting the time of onset of cancer in the first transgenicanimal; and

(3) Comparing the time of onset of cancer in the first transgenic animalto the time of onset of cancer in a second transgenic animal having aphenotype of reduced or altered ARF-p19 activity, which has not beenexposed to the composition,

wherein a statistically significant increase in the time of onset ofcancer in the first transgenic animal relative to the time of onset ofthe symptoms in the second transgenic animal indicates the potential ofthe composition for causing or exacerbating cancer.

A second method of assessing the oncogenic potential of a compositionusing the transgenic animals of the invention comprises the steps of:

(1) Administering a known dose of the composition to a first transgenicanimal having a phenotype of reduced or altered ARF-p19 activity, at aninitial time, t₀;

(2) Determining the extent of cancer in the first transgenic animal at alater time, t₁; and

(3) Comparing, at t₁, the extent of cancer in the first transgenicanimal to the extent of neurological symptoms in a second transgenicanimal having a phenotype of reduced or altered ARF-p19 activity, whichhas not been exposed to the composition at t₀,

wherein a statistically significant increase in the extent of cancer att₁ in the first transgenic animal relative to the extent of the symptomsat t₁ in the second transgenic animal indicates the potential of thecomposition for causing or exacerbating cancer.

A third method of assessing the oncogenic potential of a compositionusing the transgenic animals of the invention comprises the steps of:

(1) Administering a known dose of the composition to a first transgenicanimal having a phenotype of reduced or altered ARF-p19 activity;

(2) Measuring the lifespan of the first transgenic animal; and

(3) Comparing the lifespan of the first transgenic animal to thelifespan of a second transgenic animal having a phenotype of reduced oraltered ARF-p19 activity, which has not been exposed to the composition,

wherein a statistically significant decrease in the lifespan of thefirst transgenic animal relative to the lifespan of the secondtransgenic animal indicates the potential of the composition for causingor exacerbating cancer.

In both of the above sets of methods, the composition may comprise achemical compound administered by circulatory injection or oralingestion. The composition being evaluated may alternatively comprise apolypeptide administered by circulatory injection of an isolated orrecombinant bacterium or virus that is live or attenuated, wherein thepolypeptide is present on the surface of the bacterium or virus prior toinjection, or a polypeptide administered by circulatory injection of anisolated or recombinant bacterium or virus capable of reproductionwithin a mouse, and the polypeptide is produced within a mouse bygenetic expression of a DNA sequence encoding the polypeptide.Alternatively, the composition being evaluated may comprise one or morenucleic acids, including a gene from the human genome or a processed RNAtranscript thereof.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following examples are, therefore, to beconstrued as merely illustrative and not limitative of the remainder ofthe disclosure in any way whatsoever.

The entire text of all publications cited above and below are herebyincorporated by reference.

EXAMPLES Example 1

cDNA Sequences Encoding ARF-p19

Tandemly linked INK4A (MTS1, CDKN2) and INK4B (MTS2) genes on the shortarm of human chromosome 9 encode distinct 16 kDa and 15 kDa inhibitors(InK4a-p16 and InK4b-p15, respectively) of the G1 cyclin D-dependentkinases CDK4 and CDK6 (Serrano et al., Nature 366:704-707 (1993); Hannonand Beach, Nature 371:257-261 (1994)). Homozygous co-deletion of INK4Aand INK4B, hemizygous deletions of INK4A together with point mutationswithin the remaining allele, and de novo methylation of an CpG islandextending into exon 1 of INK4A (Merlo et al., Nature Med. 7:686-692(1995)) are commonly observed in human cancers, suggesting thatInK4a-p16, and perhaps InK4b-p15, function as tumor suppressors (Kamb,A. et al., Science 264:436440 (1994); Noburi, T. et al., Nature368:753-756 (1994); Sheaff and Roberts, Curr. Biol. 5:28-31 (1994);Hunter and Pines, Cell 79:573-582 (1995)). Two other members of the IAW4gene family, InK4c-p18 and INK4d-p19, map to different human chromosomes(Guan et al., Genes & Develop. 8:2939-2952 ((1994); Chan et al., Mol.Cell. Biol. 15:2682-2688 (1995); Hirai et al., Mol. Cell. Biol.15:2672-2681 (1995); Okuda et al., Genomics (in press) (1995)).

The human INK4A gene yields transcripts that initiate at two promoters,the first (E1α) located in close proximity to other InK4a-p16 codingexons and the second (E1β) mapping centromerically in close proximity tothe INK4b gene (Stone, S., et al., Cancer Res. 55:2988-2994 (1995); Mao,L., et al., Cancer Res. 55:2995-2997 (1995)). A long microsatellite(CA_(N)) repeat downstream of exon 1β is highly polymorphic. Thenucleotide sequence of mRNAs derived from exon 1β include a 5′ AUG codonwhich, if used to initiate protein synthesis in fully splicedtranscripts, can yield another polypeptide (here designated ARF-p19)derived from a theoretical Alternative Reading Frame that includes mostof the exon 2 coding sequences represented in INK4a-p16 mRNA. Twoclasses of transcripts (α and β, containing 5′ sequences derived fromexons 1α and 1β, respectively), although virtually identical in length,have been successfully identified by reverse transcription andpolymerase chain reactions (RT-PCR) using MRNA templates from a varietyof human tissues, but others inferred that the P transcript is unlikelyto encode a protein (Stone, S., et al., Cancer Res. 55:2988-2994 (1995);Mao, L., et al., Cancer Res. 55:2995-2997 (1995)). However, as describedherein, mouse exon 1β sequences are spliced to exon 2 of the INK4A geneto generate transcripts encoding a polypeptide that is completelydifferent in amino acid sequence from InK4a-p16.

A mouse erythroleukemia (MEL) cell DNA library (5′ Stretch λgt10,Clontech, Palo Alto, Calif.) was screened with a full-length humanInK4a-p16 probe, and twelve hybridizing cDNAs subcloned into pBluescript(Stratagene, La Jolla, Calif. were sequenced. One cDNA represented mouseInK4a-p16 (Quelle et al., Oncogene 11:635-645 (1995)) while theremaining clones, designated ARF-p19, contained alternative sequencesderived from exon 1β (FIG. 1; SEQ ID NO:1). As confirmed by Southernblotting analysis, the unrelated sequences from the 1α and 1β exonshybridized to distinct genomic DNA fragments.

The mouse β MRNA, which directs the expression of ARF-p19 in a varietyof tissues (see Examples 2 to 4), contains an AUG codon at nucleotides43-45 flanked by Kozak consensus sequences, and translation from thisinitiator would yield a 169 amino acid polypeptide of 19,349 daltons(ARF-p19; SEQ ID NO:2). Splicing of exon 1β to exon 2 of the INK4A geneoccurs at the same acceptor site as that used by exon 1β but changes theexon 2 reading frame to generate an entirely novel protein containing105 exon 2-derived residues. The mouse and human 1β exons are conservedin length, except for one additional arginine codon in the human β MRNAlocated just 5′ of the splice donor site. The reading frame (SEQ IDNO:3; FIG. 7) for the human ARF-p19 protein (SEQ ID NO:4) is only 132codons in length due to a predicted TGA terminator in place of CCA atnucleotides 436-438 (FIG. 1). Mouse and human ARF-p19 polypeptides are44% identical through their exon 1β segments and 46% identical overall.By comparison, INK4A exon 1α segments are 72% identical, with mouse andhuman InK4a-p16 proteins sharing 65% overall identity.

The ARF-p19 proteins are highly basic, as indicated by their higharginine content (human, 21% Arg; mouse 22% Arg) and are unrelated toknown proteins in searchable databases. However, several knownRNA-binding proteins, while not having amino acid sequences that are perse homologous to that of ARF-p19, nonetheless resemble ARF-p19 by havingstretches of arginine-rich sequences. Moreover, in at least someinstances, these arginine-rich regions have been implicated in thebinding of these proteins to specific RNA sequences (Craven, M. G., etal., J. Bacteriol. 176:1394-1404 (1994): Calnan, B. J., et al., Science252:1167-1171 (1991), and erratum, 255:665 (1992)); Lazinski, D., Cell59:207-218 (1989); Tao, J., et al., Proc. Natl. Acad. Sci. (U.S.A.)89:2723-2726 (1992)).

There are precedents for dual utilization of coding sequences in smallvirus genomes which cannot exceed a complexity that prevents theirpackaging into virions (Lamb and Horvath, Trends in Genetics 7:261-266(1991); Cullen, Annu. Rev. Microbiol. 45:219-250 (1991)). In contrast,eukaryotic genes are composed largely of introns and are more widelyspaced, presumably relieving them from evolutionarily imposed sizeconstraints. In Saccharonryces cerevisiae, the SNF6 locus and genesencoding certain glycolytic enzymes exhibit overlapping reading frameson opposite strands (Estruch and Carlson, Mol. Cell. Biol. 10:2544-2553(1990); Boles and Zimmermann, Mol. Ben. Genet. 243:363-368 (1994)),whereas the stress response gene, DDR48, includes two overlapping butasyntactic reading frames, each with a capacity to encode a protein of˜45 kDa (Treger and McEntee, Mol. Cell. Biol. 10:3174-3184 (1990)). Inthese cases, however, only one of the two asyntactic reading framesappears to be expressed. Recently, Labarriere et al. (J. Biol. Chem.270:19205-19208 (1995)) reported that transcripts originating from anovel promoter in the human growth hormone (GH) gene have the potentialto specify a 107 amino acid protein, the C-terminal half of which arisesfrom a second reading frame in GH exons 1 and 2. Antibodies to theC-terminus of this predicted polypeptide histochemically stained asubpopulation of pituitary cells, arguing for limited focal translationof this mRNA. In general, however, overlapping genes noted as such inthe available databases have not been assigned dual protein products.That two INK4A-coded polypeptides can each induce cell cycle arrest,albeit at different points in the cycle and via apparently distinctmechanisms (see Example 5), suggests that their unitary inheritance hasfunctional significance.

It is noteworthy that alternative splicing of transcripts from anothercell cycle-controlling gene, that encoding integrin β₁, produces asecond gene product, β_(1C), that also functions to regulate cell cyclearrest (Merdith et al., Science 269:1570-1572 (1995)). However, unlikeARF-p19 and InK4a-p16, the integrin β₁ and β_(1C) reading frames overlapsyntactically rather than a syntactically. Specifically, β_(1C) containsa carboxyl-terminal 48-amino acid sequence that replaces thecarboxyl-terminal 21 amino acids found in β₁; otherwise, however, thesequences of the two proteins are identical. If alternative splicing ofRNA transcripts is a recurring motif of cell cycle gene expression then,conceivably, a limited number of common RNA splicing factors couldaffect the expression of many proteins involved in the regulation ofprogression through the cell cycle.

Example 2

ARF-p19 is Expressed in Mouse Cells and Tissues

In a survey of mouse tissues and cells lines α and β mRNAs of similarlength (˜1 kb) were detected by Northern blotting with specific exon1αand exon 1β probes and, in agreement, by RT-PCR using specific 5′primers (FIG. 2).

For RT-PCR Analysis, polyadenylated MRNA was prepared from tissuesexcised from normal female mice (C3H/HEJ, Jackson Laboratories, BarHarbor, Me.), and cDNA was synthesized from 50 ng of polyA MRNAtemplates according to manufacturer's instructions (StrataScript RT-PCRKit, Stratagene) (Quelle et al., Oncogene 11:635-645 (1995)). PCRamplification of either mouse ARF-p19 β transcripts, or of InK4a-p16αtranscripts, was performed using a common antisense primer having thesequence

5′-GCAAAGCTTGAGGCCGGATTTAGCTCTGCTC  SEQ ID NO:5

and either an ARF-p19 specific sense primer having the sequence

5′-AGGGATCCTTGGTCACTGTGAGGATTC  SEQ ID NO:6

or an InK4a-p16 specific sense primer having the sequence

5′-CGGGATCCGCTGCAGACAGACTGGCCAG  SEQ ID NO:7

with 35 cycles of denaturation (95° C., 1 min), annealing (65° C., 45sec), and extension (720° C., 2 min). Products (˜0.5 kb, 10 μl per lane)were electrophoresed on 1.5% agarose gels and blotted onto nylonmembranes (Hybond N, Amersham, Arlington Heights, Ill.). ³²P-labeledprobes which specifically recognized exon 1β (ARF-p19, bases 1-228 ofSEQ ID NO:1), exon 1α (InK4a-p16, bases 58-182), or an antisenseoligonucleotide derived from exon 2 and having the sequence

5′-CGTCTAGAGCGTGTCCAGGAAGCCTTCC  SEQ ID NO:8

were hybridized at 50° C. in neutral pH buffer containing 0.9M NaCl andwashed in 0.015M NaCl, 0.1% SDS at the same temperature.

Specific amplification of 0 transcripts and hybridization of theproducts with exon 1β (FIG. 2(A)) and exon 2 (FIG. 2(B)) probes revealedtheir ubiquitous expression in various organs. The signal wasparticularly high using mRNA templates from MEL cells from which both αand β cDNAs have been cloned. By contrast, α mRNAs encoding INK4a-p16were expressed at relatively high levels in only few tissues (Quelle etal., Oncogene 11:635-645 (1995)), and the more restricted patterns ofhybridization observed with the exon 1α and exon 2 probes confirmed thespecificity of the PCR primers (FIGS. 2(C) and 2(D)). Importantly,amplified a transcripts generated no signal at all after hybridizationwith the exon 1β probe and vice versa, and no products were obtained inthe absence of mRNA templates (FIG. 2). ARF-p19-encoding β transcriptswere also detected in other cell lines, including CTLL-2 and RL-12 Tcells, and NFS112 B cells, but were absent from. NIH-3T3 fibroblasts andBAC1.2F5 macrophages, both of which have sustained deletions of theINK4A locus (data not shown).

Example 3

Antibodies to ARF-p19 and Detection of ARF-p19 Proteins

An antiserum directed to the unique carboxy-terminal amino acidsequences was generated using a synthetic, conjugated carboxy terminaloligopeptide derived from the ARF-p19 protein using techniquespreviously described for InK4a-p16-derived oligopeptides (Quelle et al.,Oncogene 11:635-645 (1995)). Specifically, a synthetic peptide havingthe sequence (SEQ ID NO:9):

NH₂-Val-Phe-Val-Tyr-Arg-Trp-Glu-Arg-Arg-Pro-Asp-Arg-Arg-Ala

corresponding to residues 156-169 of murine ARF-p19 protein (SEQ IDNO:2) was used.

The antibody to ARF-p19 carboxy-terminal sequences encoded by the β nRNAprecipitated a protein with an apparent molecular mass of about 22 kDa(i.e., AR-p19) after transcription and translation of the β cDNA invitro (FIG. 3, lane 8). There is no evidence that ARF-p19 undergoespost-translational modification(s) in vivo that detectably alter itsmobility on denaturing gels (see Example 4), so the apparent disparitybetween the masses of the predicted and translated proteins likelyreflect the unusual amino acid composition of ARF-p19 (FIG. 1). Anantiserum to the C-terminus of mouse InK4a-p16 detected the cognateprotein (FIG. 3, lane 6) but did not cross-react with ARF-p19 (FIG. 3,lane 9).

Sequences encoding a hemagglutinin (HA) epitope tag were added to the 5′end of the ARF-p19 cDNA by polymerase chain reaction (P)CR) using aforward primer containing a 5′ BamHI site (underlined) having thesequence (SEQ ID NO:10):

5′-CGGGATCCGAATTCAGCCATGGGTTACCATACGACG-TCCCAGACTACGCTACCGGTCGCAGGTTCTTGGTCAC

and a reverse primer extending over the single BssHHI site (underlined)in exon 1β having the sequence of the reverse complement of residues68-87 of SEQ ID NO:1, i.e.,

5′-GCCCGCGCGCTGAATCCTCA  SEQ ID NO:11

The PCR product was digested with BamHI and BssHII, subcloned into theoriginal cDNA in pBluescript, and resequenced. The resultant ARF-p19fusion protein, in which the amino terminus of ARF-p19 is tagged with ahemagglutinin (HA) epitope, has a mobility that is slightly retardedcompared to that of wild-type ARF-p19 (FIG. 3, lane 2). The HA-taggedARF-p19 protein could be detected with anti-HA serum (see below).

The endogenous ARF-p19 protein could also be detected in lysates ofmouse MEL cells (FIG. 4(A), lane 9, arrow), which synthesize high levelsof both α and β mRNAs (FIG. 2). Protein InK4a-p16, expressed in MELcells (FIG. 4(C), lane 9), was not detected with antiserum to ARF-p19,nor vice versa (compare FIGS. 4(A) and 4(C)), confirming the specificityof the antisera and indicating as well that an N-terminally truncatedInK4a-p16 protein, which would have carboxylterminal ARF-p19 sequences,does not normally arise by initiation from internal AUG codons withinthe β mRNA.

In cytospins prepared from cells harvested 48 hours after infection withviruses encoding HA-tagged ARF-p19, immunofluorescence using detectablylabeled antibodies to the ARF-p19 C-terminus (FIG. 5(A)) or to theamino-terminal HA epitope (FIG. 5(C)) demonstrate that both untagged andHA-tagged ARF-p19 localize to the cell nucleus. This reflects thestrictly nuclear localization of wild-type ARF-p19, which is normallyexpressed in derivatives of Balb/c fibroblasts (data not shown).

Example 4 Production of Recombinant ARF-p19 Native and Fusion Proteins

Baculoviral Expression in Insect Sf9 Cells

For expression in insect Sf9 cells (Kato et al., Genes & Devel.7:331-342 (1993)), an EcoRI fragment encoding tagged ARF-p19 wasinserted into the pVL1393 baculovirus vector (Pharmingen, San Diego,Calif.). Insect Sf9 cells were infected and harvested as previouslydescribed (Kato et al., Genes & Devel. 7:331-342 (1993); see alsoRichardson, C. D., ed., Baculovirus Expression Protocols, Methods inMolecular Biology Vol. 39, Walker, J. M., series ed., Humana Press,Totowa, N.J. (1995), Chapters 1-5 and 11).

When the cDNA encoding HA-tagged ARF-p19 was expressed under baculoviralcontrol in insect Sf9 cells, a protein of slightly slower mobility thanthat of the endogenous protein in MEL cells was detected with antibodyto ARF-p19 (FIG. 4(A), lane 2) or to the hemagglutinin (HA) tag (FIG.4(B), lane 2).

Virus Production and Infection of Mammalian Cells

Both untagged and HA-tagged ARF-p19 cDNAs were subcloned into the EcoRIsite of the SRa-MSV-tk-neo retroviral vector (Muller et al., Mol. Cell.Biol. 11:1785-1792 (1994)) for production of virus. Human kidney 293Tcells were transfected with 15 μg ecotropic helper virus DNA plus 15 μgSRα vector DNA using a modified calcium phosphate precipitationtechnique (Chen and Okayama, Mol. Cell. Biol. 7:2745-2752 (1987)). Cellsupernatants containing infectious retroviral pseudotypes were harvested24-60 hours post-transfection, pooled on ice, and filter (0.45μ)sterilized. Virus infections of exponentially growing mouse fibroblastsin 100 mm diameter culture dishes were performed at 37° C. in a 4% CO₂atmosphere using 2 ml virus-containing supernatants containing 8 μg/mlpolybrene (Sigma, St. Louis, Mo.). After 3 hours, 10 ml fresh medium wasadded. Cells were harvested 48 hours after infection and their DNAcontent was analyzed by flow cytometry (Matsushime et al., Cell65:701-703 (1991)).

Mammalian cell lines were maintained in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutanine,and 100 units/ml penicillin and streptomycin (Gibco, Grand Island,N.Y.). NIH-3T3 cells (Rb⁺, INK4A⁻, p53 status uncertain) weretransfected with vectors encoding D-type cyclins alone or with CDK4, andpolyclonal populations derived from pooled, drug resistant transformantswere used (Quelle et al., Genes & Devel. 7:1559-1571 (1993)).Derivatives of Balb-3T3 cells (Rb status uncertain, INK4a-p16⁺, p53⁻)were provided by G. Zambetti (St. Jude Children's Research Hospital),and the 293T retrovirus packaging line (Pear et al., Proc. Natl. Acad.Sci. USA 90:8392-8396 (1993)) was obtained from Charles Sawyers (UCLA)with permission from David Baltimore (MIT).

For analysis of ARF-p19 or InK4a-p16 expression, pelleted mammaliancells were disrupted in ice-cold cell lysis buffer (1 x 107 cells/nil)for 1 hour on ice. Nuclei and debris were removed by centrifugation in amicrofuge at 12,000 rpm for 10 min at 4° C. Supernatants were boiled ingel loading buffer, and proteins (2×10⁵ cell equivalents per lane) wereseparated on denaturing gels as above and transferred ontonitrocellulose (Quelle et al., Genes & Devel. 7:1559-1571 (1993)).Proteins were detected by enhanced chemiluminescence (ECL, Amershani)according to manufacturer's specifications with ARF-p19 or InK4a-p16antisera or with 12CA5 monoclonal antibody to the HA tag (ICN, CostaMesa, Calif.). Assays for CDC2 and CDK2-associated histone H1 kinaseactivity were performed as previously described (Matsushime et al., Cell71:323-334 (1992)). In some experiments, specifically immunoprecipitatedCDKs or cyclins were separated on denaturing gels and immunoblotted withanti-ARF-p19 as above.

Infection of INK4negative NIH-3T3 cells engineered to overexpress cyclinD1 with a retrovirus containing the β cDNA led to ectopic ARF-p19synthesis (FIG. 4A, lane 4). When the HA-tagged ARF-p19 protein wasintroduced, the polypeptide again migrated with a slightly slowermobility than the wild-type protein (FIG. 4A, lane 5 versus 4) and wasalso revealed with anti-HA serum (FIG. 4B, lane 5). Unlike NIH-3T3 cellswhich lack the INK4A gene and do not express ARF-p19 (FIG. 4A, lane 3)or InK4a-p16 (FIG. 4C, lanes 3-5), derivatives of Balb-3T3 cellssynthesize both proteins (FIGS. 4A and 4C, lanes 7 and 8). Therefore,the β transcript encodes an authentic ARF-p19 protein which iscoexpressed with InK4a-p16 in MEL and Balb-3T3derived cell lines.

Example 5

Induction of Cell Cycle Arrest by ARF-p19

In the experiments shown in FIG. 4, the DNA content of cells expressingARF-p19 was concomitantly measured in order to assess the effect(s) ofARF-p19 on the cell cycle. Surprisingly, infection of NIH-3T3 cells for48 hours with retroviruses encoding ARF-p19 induced cell cycle arrest inboth the G1 (2N DNA content) and G2/M (4N DNA content) phases of thecell cycle with a proportional loss of cells in S phase (DNA contentbetween 2N and 4N) (Table 1). Cells infected with the empty vector weredistributed throughout the cycle in a manner indistinguishable fromuninfected cells. In similar experiments performed with cells engineeredto overexpress cyclin D1, a greater proportion of ARF-p19 expressingcells arrested in G2/M versus G1 (Table 1). Proliferating cyclin D1overexpressors have a 20-30% contracted G1 phase interval and acompensatory shortening of their doubling time, so that their overallcell cycle distribution is unchanged (Quelle et al., Genes & Devel.7:1559-1571 (1993)). However, the relative increase in the G2/M versusthe G1 phase fraction suggests that cyclin D1 overexpression partiallyovercomes the p19-induced G1 block.

TABLE 1 Cell Cycle Arrest by ARF-p19 Cell Cycle Distribution Cell LineVector (48 hrs post-infection) (No. Expts) cDNA Insert % G₀G1 % S % G2/MNIH-3T3 (7) None 42.4 ± 3.3 42.0 ± 2.3 15.6 ± 3.3 NIH-3T3 (8) P19^(ARF)66.6 ± 5.2 12.5 ± 2.6 20.9 ± 5.1 3T3-D1 (8) None 41.1 ± 6.6 39.1 ± 4.419.8 ± 2.7 3T3-D1 (9) p19^(ARF) 53.0 ± 4.3 11.6 ± 2.8 35.4 ± 4.2BALB-3T3 (3) None 36.5 ± 8.9 49.0 ± 8.8 14.5 ± 1.2 BALB-3T3(3) P19^(ARF)57.1 ± 4.9 26.0 ± 2.8 16.9 ± 1.4

In these experiments, the DNA content of NIH-3T3 cells infected for 48hours with a control vector or with retroviruses encoding ARF-p19 wasdetermined by FACS analysis. Proliferating polyclonal derivativesoverexpressing cyclin D1 (3T3-D1) are not redistributed through the cellcycle, because the shortening of their G1 phase by 20-30% is compensatedby a reduced generation time (Quelle et al., Genes & Devel. 7:1559-1571(1993)). ARF-p19-induced G2→M arrest in all cyclin D overexpressors wasmore marked than in parental cells. Arrested cells were fully viable,lacked metaphases, and contained intact nuclei (FIG. 5), features allindicative of arrest in interphase.

Infection of NIH-3T3 or 3T3-D1 overexpressors with retrovirusesexpressing InK4a-p16 or InK4d-p19 leads only to G1 phase arrest (Hiraiet al., Mol. Cell. Biol. 15:2672-2681 (1995); Quelle et al., Oncogene11:635-645 (1995)). The fact that a significant proportion of cellsectopically expressing ARF-p19 accumulate in G2JM suggests that theaction of the ARF-p19 protein is not limited to effects relating toInK4a-p16 activity.

The phenotype of the ARF-p19 expressors was unusual in that many of theinfected NIH-3T3 cells appeared rounded and highly refractile,superficially similar to those in mitosis. However, cells enforced toexpress ARF-p19 exhibited only a minor (<0.2%) metaphase fractioncompared to cells infected with the vector control (˜3%). Nor did thecells undergo apoptosis, as determined by the following sensitive flowcytometric assay and by their lack of DNA fragmentation.

Apoptosis Assay

Trypsinized cell suspensions were washed and suspended in 0.5 ml PBS andpipetted dropwise into 5 ml of 1% buffered paraformaldehyde on ice withgentle mechanical mixing. After 15 min incubation on ice, cells werepelleted, washed with 10 ml cold PBS, and the pellets were resuspendedin 1 ml 70& ethanol pre-cooled at −20° C. Samples stored at −20° C.overnight were resuspended, divided into two equal aliquots, collectedby centrifugation, and washed twice with ice cold PBS. Duplicate sampleswere resuspended in 50 μl reaction mixtures containing 1× terminaldeoxynucleotidyl transferase (TdT) buffer, CoCl₂, and digoxigenin-11-UTPwith or without 0.5 μl TdT (all supplied as a TdT kit by BoehringerMannheim Corp., Indianapolis, Ill.). After 30 min incubation at 37° C.,1 ml of ice cold PBS was added, and recentrifuged cells were suspendedin 100 μl of a 1:40 dilution in PBS of anti-digoxigenin-FITC monoclonalantibody and incubated for 30 minutes in the dark at room temperature.Cells were sequentially washed in 1 ml of ice cold PBS containing 2mMsodium azide and 0.35% bovine serum albumin (BSA), and then in 1 ml 0.1%Triton X-100 in PBS, and resuspended in 1 ml PBS-azide-BSA containing 50μg/ml propidum iodide. RNAse (50 μg/ml) was added, and after 30 minuteincubation at room temperature, samples were filtered for flow cytometryand analyzed for DNA content (red PI fluorescence) and TdT-labeled DNAfragments (green FITC) on a Becton Dickinson FACScan (Matsushime et al.,1991). DNA fragmentation was quantitated by determining the differencein FITC fluorescence between duplicate samples incubated with andwithout TdT. Human Jurkat T cells treated with 100 μM etoposide for 6hrs were routinely included as positive controls for cells undergoingapoptosis.

Immunofluorescence

Cells were harvested 48 hours after infection and spun onto glass slides(5×10⁴ cells/slide) using a Scimetrics Cytospin3 at 500 rpm for 5 min.Following fixation for 10 min at room temp in 3% paraformaldehyde,slides were washed 3 times with phosphate-buffered saline (PBS),permeabilized in 0.2% Triton X-100 for 10 min at room temperature, andwashed 3 more times with PBS. After 30 min incubation in blockingsolution (PBS containing 1% dry milk), cells were incubated at roomtemperature in a humidified chamber for 1 hour with primary antibody (5μg/ml 12CAS MAb or 1:600 ARF-p19 polyclonal antiserum diluted inblocking solution). To confirm the specificity of ARF-p19 antiserum,primary antibody was incubated with ARF-p19 peptide for 1 hour attemperature prior to incubation with cells. After six washes withblocking solution, secondary antibody incubations were performed inblocking solution for 30 min, using 1:50 dilutions of eitherFITC-conjugated sheep anti-mouse or FITC conjugated donkey anti-rabbitIgG (Amersham, Arlington Heights, Ill.). After six washes with PBS,cells were stained with Hoescht dye 33258 (1 μg/ml), wet mounted withvectashield medium (Vector, Burlingame, Calif.), and photographed at600X magnification through a microscope equipped with epifluorescenceoptic (Olympus, Lake Success, N.Y.).

When cytospins were prepared from cells harvested 48 hours afterinfection with viruses encoding HA-tagged ARF-p19, immunofluorescenceperformed with antibodies to the ARF-p19 C-terminus (FIG. 5(A)) or tothe N-terminal HA epitope (FIG. 5(C)) revealed the protein in the cellnucleus. Greater than 80% of the infected cells stained brightlycompared to uninfected cells (not shown) or to those stained withpeptide blocked serum (FIG. 5(B)). No nuclear dissolution or mitoticfigures were observed, confirming that the cells arrested in interphase.

Example 6

Effects of ARF-p19 on CDK Activity

When lysates of ARF-p19-infected NIH-3T3 cells were precipitated withspecific antisera to CDC2 or CDK2 and histone H1 kinase activity wasmeasured in immune complexes, the activities of both CDKs were greatlyreduced relative to proliferating populations infected with the controlvector (data not shown). The observed ˜10-fold decrease in CDC2 kinaseis consistent with the loss of the mitotic fraction, whereas the ˜5 folddrop in CDK2 activity likely reflects a redistribution of cells from Sphase into G1 and G2. Indeed, given the dissimilarity in structurebetween ARF-p19 and known CDK inhibitors, it seems unlikely that theprotein interacts directly with CDKs or cyclins. In agreement, whenCDC2, CDK2, CDK4, CDK6, cyclins D1, D2, E, and A immunoprecipitates fromNML cell lysates were blotted with anti-ARF-p19, no coprecipitation ofARF-p19 was observed. Reconstruction experiments with baculovirusexpression vectors in insect Sf9 cells also failed to provide convincingevidence for associations between ARF-p19 and these cell cycleregulators. The mechanism(s) by which ARF-p19 induces cell cycle arrestremains unclear, although the propensity of some cells to arrest in G2argues that these ARF-p19-induced effects are pRb-independent.

Example 7

ARF-p19 Mutations in Human Cancers

Current evidence indicates that InK4a-p16 functions upstream of cyclinD-dependent kinases and pRb in a biochemical pathway that regulates exitof G1 phase cells into S phase. InK4a-p16 cannot induce G1 arrest incells that lack pRb function (Guan et al., Genes Devel. 8:2932-2952(1994); Tam et al., Oncogene 9:2663-2674 (1994); Lukas et al., Nature375:503-506 (1995); Medema et al., Proc. Natl. Acad. Sci. USA92:6289-6293 (1995); Koh et al., Nature 375:506-510 (1995)), and in lungcancer, INK4A deletions are restricted to tumors that retain pRbactivity and vice versa, implying that a loss of either gene makescompromise of the other irrelevant (Otterson et al., Oncogene9:3375-3378 (1994)). Increased levels of InK4a-p16 are generallyobserved in pRb-negative tumor cells (Serrano et al., Nature 366:704-707(1993); Bates et al., Oncogene 9:1633-1640 (1994); Lukas et al., J.Cell. Biol. 125:625-638 (1994); Tam et al., Oncogene 9:2663-2674(1994)), suggesting that InK4a-p16 expression may be somehow governed bypRb itself or, alternatively, that pRb provides a predisposing selectivepressure that favors elimination of pRb-mediated controls (Sherr andRoberts, Genes Devel. 9:1149-1163 (1995)).

Of the numerous InK4a-p16 mutations found in human cancers, only a fewhave been experimentally evaluated for effects on the cell cycle.However, two which clearly abrogate InK4a-p16 inhibitory functions (R87βand H98β in Koh et al., Nature 375:506-510 (1995)) are silent withregard to ARF-p19, and another (P114L in Lukas et al., Nature375:503-506 (1995)) falls outside the region of overlap betweenInK4a-p16 and ARF-p19, indicating that the latter is not a target ofinactivating mutations in these cases.

Nevertheless, most mutations involving INK4A in cancer cells fall withinthe 5′ half of exon 2, raising the possibility that some may duallyaffect InK4a-p16 and ARF-p19 or, conceivably, ARF-p19 alone. About 60%of mutations in InK4a-p16 cluster within the region overlapping ARF-p19(Hirama and Koeffler, Blood 86:841-854 (1995)), and more than 80% ofthese affect ARF-p19 primary structure. FIG. 6 shows the predictedARF-p19 mutations within this segment, compiled from data obtained withprimary tumors, xenografts, and established cell lines (Kamb, A. et al.,Science 264:436440 (1994a), Nature Genet 8:22-26 (1994b); Caldas et al.,Nature Genet. 8:27-32 (1994); Hussussian et al., Nature Genet. 8:15-21(1994); Ohta et al., Cancer Res 54:5269-5272 (1994); Zhang et al.,Cancer Res 54:5050-5053 (1994); Mori et al., Cancer Res. 54:3396-3397(1994); Hayashi et al., Biochem. Biophys. Res. Commun. 202:1426-1430(1994)). Of 50 missense and frame shift mutations, 39 involve codonsconserved between human and mouse ARF-p19 (residues in bold type) andfour (marked by asterisks) are silent in InK4a-p16. The most frequentlymutated ARF-p19 residues in sporadic cancers are Gly-69, Pro-94, Arg-98,each of which is conserved in humans and mice, and the most commondisease-related alteration in melanoma kindreds (Hussussian et al.,Nature Genet. 8:15-21 (1994); Kainb et al., Nature Genet. 8:22-26(1994b)) converts conserved Arg-115 of ARF-p19 to Leu. A furthercomplication is that frame shift mutations have the potential to producechimeric proteins. For example, those involving ARF-p19 Gln-70 (Hayashiet al., Biochem. Biophys. Res Commun. 202:1426-1430 (1994)) and Gly-74(Ohta et al., Cancer Res. 54:5269-5272 (1994)) should result in INK4A αtranscripts encoding InK4a-p16/ARF-p19 fusions in which the majority ofexon-2 sequences encode ARF-p19 residues. Conversely, an ARF-p19 frameshift involving Gly-102 (Hayashi et al., Biochem. Biophys. Res Commun.202:1426-1430 (1994)) would yield a β transcript encoding the C-terminalhalf of InK4a-p16. To the extent that mutations in ARF-p19 contribute toaberrant growth control and tumorigenesis, detection and analysis ofARF-p19-specific nucleic acids (Example 2) in a mammal serves todiagnose, or assist in the diagnosis of, existing tumors in the mammal,or to predict the mammal's predisposition for developing certain formsof cancer.

12 713 base pairs nucleic acid double unknown DNA (genomic) CDS 43..548CDS 43..551 1 GTCACAGTGA GGCCGCCGCT GAGGGAGTAC AGCAGCGGGA GC ATG GGT CGCAGG 54 Met Gly Arg Arg 1 TTC TTG GTG ACT GTG AGG ATT CAG CGC GCG GGC CGCCCA CTC CAA GAG 102 Phe Leu Val Thr Val Arg Ile Gln Arg Ala Gly Arg ProLeu Gln Glu 5 10 15 20 AGG GTT TTC TTG GTG AAG TTC GTG CGA TCC CGG AGACCC AGG ACA GCG 150 Arg Val Phe Leu Val Lys Phe Val Arg Ser Arg Arg ProArg Thr Ala 25 30 35 AGC TGC GCT CTG GCT TTC GTG AAC ATG TTG TTG AGG CTAGAG AGG ATC 198 Ser Cys Ala Leu Ala Phe Val Asn Met Leu Leu Arg Leu GluArg Ile 40 45 50 TTG AGA AGA GGG CCG CAC CGG AAT CCT GGA CCA GGT GAT GATGAT GGG 246 Leu Arg Arg Gly Pro His Arg Asn Pro Gly Pro Gly Asp Asp AspGly 55 60 65 CAA CGT TCA CGT AGC AGC TCT TCT GCT CAA CTA CGG TGC AGA TTCGAA 294 Gln Arg Ser Arg Ser Ser Ser Ser Ala Gln Leu Arg Cys Arg Phe Glu70 75 80 CTG CGA GGA CCC CAC TAC CTT CTC CCG CCC GGT GCA CGA CGC AGC GCG342 Leu Arg Gly Pro His Tyr Leu Leu Pro Pro Gly Ala Arg Arg Ser Ala 8590 95 100 GGA AGG CTT CCT GGA CAC GCT GGT GGT GCT GCA CGG GTC AGG GGCTCG 390 Gly Arg Leu Pro Gly His Ala Gly Gly Ala Ala Arg Val Arg Gly Ser105 110 115 GCT GGA TGT GCG CGA TGC CTG GGG TCG CCT GCC GCT CGA CTT GGCCCA 438 Ala Gly Cys Ala Arg Cys Leu Gly Ser Pro Ala Ala Arg Leu Gly Pro120 125 130 AGA GCG GGG ACA TCA AGA CAT CGT GCG ATA TTT GCG TTC CGC TGGGTG 486 Arg Ala Gly Thr Ser Arg His Arg Ala Ile Phe Ala Phe Arg Trp Val135 140 145 CTC TTT GTG TTC CGC TGG GTG GTC TTT GTG TAC CGC TGG GAA CGTCGC 534 Leu Phe Val Phe Arg Trp Val Val Phe Val Tyr Arg Trp Glu Arg Arg150 155 160 CCA GAC CGA CGG GCA TAG CTTCAGCTC AAGCACGCCC AGGGCCCTGG 581Pro Asp Arg Arg Ala 165 AACTTCGCGG CCAATCCCAA GAGCAGAGCT AAATCCGGCCTCAGCCCGCC TTTTTCTTCT 641 TAGCTTCACT TCTAGCGATG CTAGCGTGTC TAGCATGTGGCTTTAAAAAA TACATAATAA 701 TGCTTTTTTT TT 713 169 amino acids amino acidlinear protein 2 Met Gly Arg Arg Phe Leu Val Thr Val Arg Ile Gln Arg AlaGly Arg 1 5 10 15 Pro Leu Gln Glu Arg Val Phe Leu Val Lys Phe Val ArgSer Arg Arg 20 25 30 Pro Arg Thr Ala Ser Cys Ala Leu Ala Phe Val Asn MetLeu Leu Arg 35 40 45 Leu Glu Arg Ile Leu Arg Arg Gly Pro His Arg Asn ProGly Pro Gly 50 55 60 Asp Asp Asp Gly Gln Arg Ser Arg Ser Ser Ser Ser AlaGln Leu Arg 65 70 75 80 Cys Arg Phe Glu Leu Arg Gly Pro His Tyr Leu LeuPro Pro Gly Asp 85 90 95 Arg Arg Ser Ala Gly Arg Leu Pro Gly His Ala GlyGly Ala Ala Arg 100 105 110 Val Arg Gly Ser Ala Gly Cys Ala Arg Cys LeuGly Ser Pro Ala Ala 115 120 125 Arg Leu Gly Pro Arg Ala Gly Thr Ser ArgHis Arg Ala Ile Phe Ala 130 135 140 Phe Arg Trp Val Leu Phe Val Phe ArgTrp Val Val Phe Val Tyr Arg 145 150 155 160 Trp Glu Arg Arg Pro Asp ArgArg Ala 165 540 base pairs nucleic acid double linear DNA (genomic) CDS142..540 3 CGCGCCTGCG GGGCGGAGAT GGGCAGGGGG CGGTGCGTGG GTCCCAGTCTGCAGTTAAGG 60 GGGCAGGAGT GGCGCTGCTC ACCTCTGGTG CCAAAGGGCG GCGCAGCGGCTGCCGAGCTC 120 GGCCCTGGAG GCGGCGAGAA C ATG GTG CGC AGG TTC TTG GTG ACCCTC CGG 171 Met Val Arg Arg Phe Leu Val Thr Leu Arg 1 5 10 ATT CGG CGCGCG TGC GGC CCG CCG CGA GTG AGG GTT TTC GTG GTT CAC 219 Ile Arg Arg AlaCys Gly Pro Pro Arg Val Arg Val Phe Val Val His 15 20 25 ATC CCG CGG CTCACG GGG GAG TGG GCA GCG CCA GGG GCG CCC GCC GCT 267 Ile Pro Arg Leu ThrGly Glu Trp Ala Ala Pro Gly Ala Pro Ala Ala 30 35 40 GTG GCC CTC GTG CTGATG CTA CTG AGG AGC CAG CGT CTA GGG CAG CAG 315 Val Ala Leu Val Leu MetLeu Leu Arg Ser Gln Arg Leu Gly Gln Gln 45 50 55 CCG CTT CCT AGA AGA CCAGGT CAT GAT GAT GGG CAG CGC CCG AGT GGC 363 Pro Leu Pro Arg Arg Pro GlyHis Asp Asp Gly Gln Arg Pro Ser Gly 60 65 70 GGA GCT GCT GCT GCT CCA CGGCGC GGA GCC CAA CTG CGC CGA CCC CGC 411 Gly Ala Ala Ala Ala Pro Arg ArgGly Ala Gln Leu Arg Arg Pro Arg 75 80 85 90 CAC TCT CAC CCG ACC CGT GCACGA CGC TGC CCG GGA GGG CTT CCT GGA 459 His Ser His Pro Thr Arg Ala ArgArg Cys Pro Gly Gly Leu Pro Gly 95 100 105 CAC GCT GGT GGT GCT GCA CCGGGC CGG GGC GCG GCT GGA CGT GCG CGA 507 His Ala Gly Gly Ala Ala Pro GlyArg Gly Ala Ala Gly Arg Ala Arg 110 115 120 TGC CTG GGG CCG TCT GCC CGTGGA CCT GGC TGA 540 Cys Leu Gly Pro Ser Ala Arg Gly Pro Gly 125 130 132amino acids amino acid linear protein 4 Met Val Arg Arg Phe Leu Val ThrLeu Arg Ile Arg Arg Ala Cys Gly 1 5 10 15 Pro Pro Arg Val Arg Val PheVal Val His Ile Pro Arg Leu Thr Gly 20 25 30 Glu Trp Ala Ala Pro Gly AlaPro Ala Ala Val Ala Leu Val Leu Met 35 40 45 Leu Leu Arg Ser Gln Arg LeuGly Gln Gln Pro Leu Pro Arg Arg Pro 50 55 60 Gly His Asp Asp Gly Gln ArgPro Ser Gly Gly Ala Ala Ala Ala Pro 65 70 75 80 Arg Arg Gly Ala Gln LeuArg Arg Pro Arg His Ser His Pro Thr Arg 85 90 95 Ala Arg Arg Cys Pro GlyGly Leu Pro Gly His Ala Gly Gly Ala Ala 100 105 110 Pro Gly Arg Gly AlaAla Gly Arg Ala Arg Cys Leu Gly Pro Ser Ala 115 120 125 Arg Gly Pro Gly130 125 amino acids amino acid linear protein 5 Met Met Met Gly Asn ValHis Val Ala Ala Leu Leu Leu Asn Tyr Gly 1 5 10 15 Ala Asp Ser Asn CysGlu Asp Pro Thr Thr Phe Ser Arg Pro Val His 20 25 30 Asp Ala Ala Arg GluGly Phe Leu Asp Thr Leu Val Val Leu His Gly 35 40 45 Ser Gly Ala Arg LeuAsp Val Arg Asp Ala Trp Gly Arg Leu Pro Leu 50 55 60 Asp Leu Ala Gln GluArg Gly His Gln Asp Ile Val Arg Tyr Leu Arg 65 70 75 80 Ser Ala Gly CysSer Leu Cys Ser Ala Gly Trp Ser Leu Cys Thr Ala 85 90 95 Gly Asn Val AlaGln Thr Asp Gly His Ser Phe Ser Ser Ser Thr Pro 100 105 110 Arg Ala LeuGlu Leu Arg Gly Gln Ser Gln Glu Gln Ser 115 120 125 31 base pairsnucleic acid single linear other nucleic acid /desc = “Synthetic DNA” 6GCAAAGCTTG AGGCCGGATT TAGCTCTGCT C 31 27 base pairs nucleic acid singlelinear other nucleic acid /desc = “Synthetic DNA” 7 AGGGATCCTTGGTCACTGTG AGGATTC 27 28 base pairs nucleic acid single linear othernucleic acid /desc = “Synthetic DNA” 8 CGGGATCCGC TGCAGACAGA CTGGCCAG 2828 base pairs nucleic acid single linear other nucleic acid /desc =“Synthetic DNA” 9 CGTCTAGAGC GTGTCCAGGA AGCCTTCC 28 14 amino acids aminoacid single Not Relevant peptide 10 Val Phe Val Tyr Arg Trp Glu Arg ArgPro Asp Arg Arg Ala 1 5 10 74 base pairs nucleic acid single linearother nucleic acid /desc = “Synthetic DNA” 11 CGGGATCCGA ATTCAGCCATGGGTTACCCA TACGACGTCC CAGACTACGC TACCGGTCGC 60 AGGTTCTTGG TCAC 74 20base pairs nucleic acid single linear other nucleic acid /desc =“Synthetic DNA” 12 GCCCGCGCGC TGAATCCTCA 20

What is claimed is:
 1. An isolated antibody that binds to an ARF-p19polypeptide, wherein said ARF-p19 polypeptide is a human ARF-p19polypeptide comprising the amino acid sequence of SEQ ID NO:4.
 2. Theisolated antibody of claim 1, wherein the antibody is generated with animmunoreactive peptide derived from SEQ ID NO:4.
 3. The isolatedantibody of claim 2, wherein the antibody is generated with a peptidecomprising 5 to 100 contiguous amino acids of SEQ ID NO:4.
 4. Theisolated antibody of claim 3, wherein the immunoreactive peptide isprepared by organic synthesis.
 5. The isolated antibody of claim 2,wherein the antibody is generated with a fusion peptide comprising 5 to100 contiguous amino acids of SEQ ID NO:4.
 6. The isolated antibody ofclaim 3, wherein the antibody is generated with a peptide comprising 5contiguous amino acids of amino acid residues 1 to 65 of SEQ ID NO:4. 7.The isolated antibody of claim 1 which is labeled.
 8. The isolatedantibody of claim 1 which is a monoclonal antibody.
 9. The isolatedantibody of claim 1 which is a polyclonal antibody.
 10. The isolatedantibody of claim 3, wherein said peptide is conjugated to a carrier.